Method for constructing of recombinant plasmid dna, encoding enzyme deacet-oxycephalosporine c- synt

专利摘要:
The present invention relates to DNA compounds and expression vectors that encode deacetoxycephalosporin C synthetase (DAOCS) and deacetylcephalosporin C synthetase (DACS) activities. The compounds can be used to construct recombinant DNA expression vectors for a wide variety of host cells, including E. coli, Penicillium, and Cephalosporium. The invention also relates to the regulatory elements of the Cephalosporium acremonium DACS/DAOCS gene. A method for selecting Penicillium transformants, including those able to synthesize cephalosporin antibiotic due to the presence of the DACS/DAOCS-encoding DNA, is also provided. This transformation system utilizes an acetamidase gene for purposes of selecting transformants.
公开号:SU1739856A3
申请号:SU884355306
申请日:1988-03-03
公开日:1992-06-07
发明作者:Доминик Инголиа Томас；Виатт Квинер Стефен；Мэри Сэмсон Сьюллен；Латер Скэтрад Пол
申请人:Эли Лилли Энд Компани (Фирма)；
IPC主号:

专利说明:

The invention relates to genetic engineering, the preparation of plasmid DNAs encoding the enzyme -C-deacetoxycephalosporin C synthetase activity (DAOSC) and deacetylcephalosporin C synthetase activity (DACS) „.
DAOCS catalyzes the reaction in which penicillin N is formed (DAOS), and is expansion, UACS catalyzes the formation of deacetylphalosporin (DAS) and DACS by hydroxylation. These reactions are the main stages of the biosynthesis of important antibiotics, such as cephalosporins from Cephalosporium acremo-, niuffl and 7 ot-methoxycephalosporins from Streptomyces clavuligerus.
The DNA fragment encoding the activity of DACS / DAOCS was introduced from the genomic DNA of Cephalosporium acremonium and
used to construct expression vectors that transform Escherichia coli strains.
Raw cell extracts from transformed E. coli exhibit DAOCS / DACS activity without any prior activation. Designed vectors and transformants provide efficient production of large quantities of the active enzyme DAOCS / DACS. The DAOCS / DACS enzyme can be used to produce DAOCS and DACS and to hydroxylate cephalosporins to form new antibiotics.
Recombinant plasmid DNAs can be used to create Penicillium, fCephalosporium strains producing penicillin and cephalosporin antibiotics.
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In the present invention, an improved method of Penicillium transformation with recombinant DNA is described. This new transformation system uses the amds gene of Aspergillus nidulans. Using acetamide (CHjCONH2) as the only carbon or nitrogen source on the transformation plates, Penicillium host cells transformed with amds gene from non-transformed cells that cannot grow on these plates due to the fact that PeniciIumum host cells are selected wild type do not express acetamidase activity
The proposed recombinant plasmid DNAs can be used to produce strains of Paecilomyces and Streptomyces, in particular S. clavulatory, which express DAOCS / / DACS. Although DNA encoding DACS / / DAOCS is isolated from Cephalosporium acremoniura, plasmid DNA containing this gene can be used to construct DACS / DAOCS expression vectors in a wide variety of host cells. Most organisms that produce penicillins and cephalosporins use the common precursor penicillin N, a substrate for DAOCS. DNA fragments encoding DACS / DAOCS can be used in
methods for constructing expression vectors in the transformation of a wide variety of organisms that produce penicillin and cephalosporin antibiotics.
DNAs encoding DACS / DAOCS are isolated with regulatory sequences that control transcription. New regulatory sequences have been described that can be used in new methods to ensure the translation and transcription of any gene product in Cephaloscorium. These regulatory sequences are particularly effective in C. acremonium.
Regulatory signals of the DACS / DAOCS gene located at the 3-terminus of the Cephalosporium acremonium DACS / DAOCS coding gene are described. These 3 -regulatory sequences encode transcription termination and RNA polyapenylation, as well as the DACS / DAOCS gene processing signals. Placing these signals at the 3'-end of the gene enhances the expression of the gene in Cephalosporium
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The DNA sequence of the Brotzu Cephalosporium acremonium strain, encoding both DACS activity and DAOCS activity, is presented as:
U20 30 40
. 5 -A AGC TTG TAG GGA GAA TTA AGG CTT GCA CGA TTC CAT GGC GGT CTC
50 60 7080 90
GAC GAT GAG GGA CCA TGC ACG ATA CAT ATT CTC CTG CGA ACC AAG
„А„ 10 ° no 120. 130 GAG AAG AGA ACT CGA TGG CTT CTT ATG ATT CGT TGA CAA AAC TTC ACA
 160 170 iftn ion AGA CAC TCG TGG GTT TAG AAT GCT ACA TTG ACG TGT GCG GCC AAG GCT
200 210 220 9chp GAG ™ MO SACIS GIC ACT TAC OGC IAA GIA GCA GIT TAA AAA
GCA GIT CCT COG CGT AAG CTA CGA GGT GOG OY ° TOA GAT ATA
CCO with tsSA CAA SsGTTS GIT StLSt "CO PBX% IOA AIC ™ w C CTC TTO CAG AAC ITT CCT CCA CGC ° TAC TCC TcfL GIC" Jg
CAA AAC AIC ACT ICC MO GTC CCC GTc V COT
№T THR SER tYS VAl PRO S Ј5 W SS
517398566
5 10
440 450 460 470
GAC GAC CTC AAG AGO GGC AAG GTC CTC ACC GAG CAC ASC GTC
152025
480 490 500 510 520 ACC ACC AAG GGT PBX TTC TAC TTG ACC GAG AGC GGC CTG GTC GAC GAC THR THR
530 540 550 560 570 GAC CAC ACG TCG GCG CGT GAG ACG TGC GTG ACC TTC ATP C ASC A T A G A G A C A A C A A A A A A A A A A C A A A C A A A C A A C A A C A C A C A C A C A C J A C A C J A C A C A C A C A C A C A C A C A C V A C A C A C A C W
455055
580 590 600 610 620 AGC GAC GAG GAG AAG AGG GCC GTG ACC CTC GAC CAC GC CGT C AAC GCC
606570
630 640 650 660 670 CGC GGC GTC GTC ACC GAG ACC GCC ACC GAG ACC GAC ACG
680 690 700 710
ACG GGC AAG TAC TCG GAC TAC TCG ACG TGC TAC TCC ATG GGC ATC GGC THR GLY LYS TYR SER ASP TYR SER THR CYS TYR SER MET GLY ILE GLY 95100105
720 730 740 750 760 GGC AAC CTG TTC CCG AAC CGG GGC TTC GAC GAC GTC TAC CAG CAC GAC TAC GLY ASU LEU PHE PRO ASN ARG GLY PHE GLU ASP VAL TRP GLN ASP TYR
110115120
770 780 790 800 810 TTC GAC CGC ATC TAC GGC GCA GCC AAG GAT GTC GCG CGC GCC GTC GCC GCTG CG CG ASC A T A A A A A A A A A A A A T C S
820 830 840 850 860 AAC TCT GTG GGC GCC GTC GTC GTC GTC GAC GAC ATC GTC GTC GTC GTC ATC AST CLE GAT
870 880 890 900 910 GAC TGC GAT CTC CTC CGC CTA CTA CGG TAC CTCA GA GTG CGGGGGGGGGGGGGGGGGGGGGGY
920 930 940 950
GAC CGC GAC GAC GAC GAC GAC GAC GA GAC GA GAC GAC GAC GAC ASCG ASC ASCG ASC ASC ASC ASC
175180185
960 970 980 990 1000 CTA TCG ACC ATC ACG CTC GTG CAC CAG ACA GCC TGC GCC AAC GGC TTC LEU SER THR ILE THR ALA CYS ALA ASN GLY PHE 190195200
-7 7398568
1010 1020 1030 1040 1050
GTG AGC CTG CAG TGC GAG GTG GAG GGA GAATTC GTC GAC CTC CCG ACG
VAL SER LEU GLN CYS GLU VAL ASP GLY GLUPHE VAL ASP LEU PRO THR
205210215
1060 1070 1080 1090 1100
CTC CCC GGC GCC ATG GTC GTC TGC GGC GGC GTC ACC CTG GCC ACC CTG GCC LEU PRO GLY ALA VAL VAL VAL PHY CYS GLY ALA VAL GLY THR LEU ALA 220 225230
1110 1120 1130 1140115C
ACG GGC GGC AAG GTC.AAG GCG CCC AAG CAC CGG GTC AAG TCT CCCGGG
ALA PRO PROTES
235,240 245250
1160. 1170 1180 1190
CGC GAC CAG CGC GTC GGC AGC AGC CGC ACG TCG AGC GTC TTC TTCCTG
ARG ASP GLN ARG VAL GLY SER SER ARG THR SER SER VAL PHE PHELEU 255 260 265
1200 1210 1220 1230 1240
CGG CCG AAG CCC GAC TTCAGC TTC AAC GTG CAG CAG TCG AGO GAG TGG
ARG PRO LYS PRO ASP PHESER PHE ASN VAL GLN SER ARG GLU TRP
270275280
1250 1260 1270 - 1280 1290
GGT TTC AAC GTC CGC ATSCCG TCG GAG CGC ACG ACG TTC AGG GAG TGG
JLY PHE ASN VAL ARG ILEPRO SER GLU ARG THR THR PHE ARG GLU TRP 285290295
1300 1310 1320 1330 1340
CTT GGC GGG AACTAT GTC AAC ATG CGG AGG GAT AAG CCG GCG GCA GCG
LEU GLY GLY ASNTYR VAL ASN MET ARG ARG ASP LYS PRO ALA. ALA ALA 300305310
1350 1360 1370 1380 1390
GAG GCG GCT GTCC GCC GCT GCC GTC
GLU ALA ALA VALPRO ALA ALA ALA PRO VAL SER THR ALA ALA PRO ILE 315320325330
1400 1410 1420 1430
GCC ACT TAG GGA ACC CGC CGA TCG ACT AAT AAA TCT ACG GGA GTT TAA ALA THR
1440 1450 1460 1470 1480 GAA GAA AAA TTG CCC TAT AAA TTG CTA DAT TTT TAA AAC ACA AAG CAT
1490 1500 1510 .. GAG TGT CAA GAG TTT CAA GTT TCA A-3
DNA fragments encoding an enzyme with DACS / DAOCS activity are isolated from the strain of Cephalosporium acremonium, known as the Brotzu strain, which is contained in the American type culture collection, Rockville, Maryland (stored under ATCC number 11550). The genomic DNA library of the Brotzu strain is constructed in the bifunctional cosmid vector pKC462A (existing in E. coli K12 under the number NRRLB-15973) and examined for the presence of sequences homologous to a number of 32 different deoxyribo oligonucleotides. This series of deoxyribo oligonucleotides was built on the basis of information on the partial amino acid sequence of the enzyme C. acremonium DACS / / DAOCS and knowledge of the genetic code. Clones homologous to one or more of 32 different deoxyribo oligonucleotides were selected.
Plasmid DNAs encoding the DACS / DAOCS enzyme are identified, from which an L / 7 kV Bam HI fragment, including the DACS / DAOCS gene, is isolated and inserted into the commercially available plasmid sC8. A plasmid which is transformed into E. coli Kt2 JA 221 is obtained. Transformants E. coli K12 JA 221 / pIT508 are deposited in the culture collection of the Northern Regional Research Laboratory (NRRu), Peori, IL 61604, under the number NRR LB-18170.
The plasmid p1T503 serves as a starting material for constructing other expression vectors. These vectors are suitable for producing the DACS / DAOCS enzyme in a recombinant host cell. For this, transformation of host cells of the recombinant plasmid DNA is carried out, which includes: a promoter and a sequence that activates the translation process; a DNA sequence that encodes a DACS / / DAOCS and is operably linked to said promoter; cultivation of the transformed recipient cell transformed under conditions that ensure the synthesis of the enzyme.

The plasmid was isolated from E. coli K12 JA 221, used to construct DACS / DAOCS expression vectors. The plasmid is used as a starting material for the construction of a plasmid, which provides a high degree of
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expression of the enzyme DACS / DAOCS in E.coli.
The construction of the plasmid includes the linking of a 2.2 kb Sst I - Bam HI fragment, encoding a DACS / / DAOCS plasmid with a 5.8 kV Nde I - Ban HI fragment of the pCZR336 plasmid, and the synthesized fragment p. Sst I - Nde I.
Plasmid pCZR336 includes a promoter derived from Dr, a sequence that activates translation, a gene operator, the first functional unit of heredity
5 (Cistron) encoding a small
peptide, and the DNA sequence encoding the hGH0 derivative At a low temperature of 30 ° C, protein copied on plasmids pCZR33C and is active and able to suppress the activity of the Dryb promoter, but when the temperature rises up to 42 ° C, the protein is inactivated and the promoter accelerates the transcription of a large amount of mRNA encoding hGH (pCZR336) or DACS / DAOCS (pIT507).
Plasmid I includes the same - | the first cistron gene, promoter
O and the sequence that activates translation as plasmid pCZR336, but includes the coding sequence of the DACS / DOACS gene from the plasmid instead of the coding
5 hGH sequences. Restrictive fragment 2.2 p. Sst I - Baga HI plasmid contains most of the DACS / DAOCS coding sequence, the rest of this coding sequence is contained in the synthesized fragment of 60 bp. Nde I - Sst I with a slight modification compared to the wild-type DNA sequence, which is about 5 (facilitates subsequent work. 5I-CATATG-3I sequence
, ШМ1,
3 -GTATAC-5 0 includes
5-ATG-3 HI, Z-TAS-5,
which encodes the amino terminal methionyl group DACS / DAOCS. The plasmid is constructed so that the promoter pb and sequences that activate translation are located,
ensuring the expression of the DNA encoding DACS / DAOCSc
At a temperature of 42tfC E.coli K12 I ii 109 / pIT507. DACS / j / DAOCS is expressed (15% of the total cell tree). The crude cell extracts of these E.coli K12 JM 109 / pI507 (transformant) are capable of catalyzing the conversion of penicillin N to DAOS DAS, while the cell extracts from non-transformed E. coli K12 JM 109 cells cannot catalyze this conversion.
Many strains of E. coli K 1.2. Possess activity of endogenous cephalosporinins encoded by the ampC locus. In this case, the DAOCS / DACS polypeptide is partially purified so that the optimum activity of DAOCS / DACS is achieved. For this purpose, the enzyme is sufficiently purified by DEAE-trivacryl to separate the endogenous E .coli cephalosporinase from the enzyme DAOCS / DACS. To eliminate the harmful effects of cephalosporinase, a strain defective in this enzyme is used. One such E.coli strain K12 A85 892 is stored at the Northern Regional Research Center under the number NRRLB-18096.
Plasmids and efficiently produce large amounts of DACS / DAOCS in E. coli. E. coli pIT507 produces DACS / DAOCS in an amount approaching 15% of the total cellular protein. Since E. coli cultivation is less complex, E. coli pI507 transformants can be used in the DACS / DAOCS production method more efficiently and more efficiently. economically than natural producers of DACS / DAOCS.
DAOCS can be used to produce DAOCS from penicillin N in a cell-free system. DAOS is not only a useful antibiotic, but can also be used as a starting material for the production of important antibiotics such as cephalexin and other cephalosporins. DACS / DAOCS is used to transform penicillins other than penicillin N into new cephalospore-4-ryne derivatives.
Cell-free extracts of penicillin and cephalosporin-forming organisms can be used to synthesize non-naturally occurring | 3-lactams. Offered
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E.coli expression vectors can be used in the DACS / DAOCS production method to transform penicillins, which are not naturally found in nature, to form new antibiotics or structures with an antibiotic core.
Plaemid is suitable for obtaining DACS / DAOCS in Eccoli not only because of the high levels of expression of the enzyme in these cells and because of the presence of a marker present on this plasmid. Many vectors encode / 5-lactamae and cells containing this vector, and can grow in the presence of certain / 3-lactam antibiotics, such as ampicillin. However, if you want to use free
Q from cells is an extract containing DACS / / DAOCS for the purpose of generating 3-lactams, then an extract that contains K-lactamase activity is undesirable. Thus, the plasmid does not encode β-lacta 5 Mazu, but uses a tetracycline-resistant gene that encodes a protein that does not react with δ-lactams. The cysteine group at position 100 is replaced with episodes in the DACS / DAOCS coding region of the plasmid of this expression vector. This conventional method can be used to replace any group with any of the other 19 naturally coded am0s.
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but acid
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Splicing in conditions
The vitro mutant genes created in this way allow the production of proteins with combinations of mutations. Such a mutant DACS / DAOCS obtained using.
A change in the amount of genetic information (thus causing the insertion or deletion of amino acids in the resulting protein) in the DNA DACS / DAOCS coding sequence changes the correspondingly encoded amino acid sequence.
There are a number of plasmids that increase the intracellular concentration of a transformed DACS / DAOCS enzyme cell containing: DNA encoding a DACS / DAOCS enzyme; a promoter and sequence that activates translation, operatively linked to the DAOC / DAOS gene. Resistant
5, transformants can only be obtained if the vector is reproduced either as an extrachromosomal element or integrated into the genomic DNA in the host cell. The plasmids described also include a gene that gives an antibiotic, or some other element that provides a means of selecting host cells that contain the vector, but such elements may not be available when the vector is integrated into the chromosomal DNA of the host cell.
Expression vectors in Penicillium and Cephalosporium are described as follows.
Plasmid pPS56 is the Cephalosporiunu lazmid pPS55 expression vector is an intermediate in the construction of plasmid pPS56. Plasmid pPS55 was constructed from the Hind III fragment of the plasmid MLC12, 3 kb. The hind III fragment of the plasmid pPS34, which includes the C promoter, acremonium IPS, fused to the coding sequence of hydrohromycin B phosphotransferase. The Bam HI 7 kb fragment containing the DACS / / DAOCS gene of the plasmid was fused with the pam 55 fragment partially treated with Bam HI, forming the plasmid pPS 56.
The Cephalosporium acremonium promoter and the sequence that activates translation on the plasmid are located in such a way that they cause expression of the DNA encoding the enzyme with the activity of the DACS / DAOCS synthetase, since during the construction of the plasmid there are no deletions or insertions acting on the promoter inconsistency activating the translation, not inserted into DNA bordering the 51 end.
The Penicillium IPS gene can be inserted into highly producing Penicillium strains in order to increase the titer of penicillins produced during fermentation.
Plasmids p3SR2, pPS51, pPS52, pPS54 and pPS61 contain the amds gene and Penicillium cells containing these plasmids, and can be selected using acetamide “Plasmids PPS57 and pPS62 contain a hybrid gene that confers resistance to hydromycin, which uses plasmids pPS59, resulting in get the plasmid pPS61. Plasmid pPS61 is designed to express Penicillium DACS / DAOCS enzyme, especially P. chrysogenum.
Plasmid pPS51 is used as a source of acetamide gene in other plasmid constructs. Plasmid pPS52 contains the DACS / DAOCS gene
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on the fragment l / 7 m. Bam HI (from plasmid pPS51) and synthesized binding molecule. Plasmid pPS52 provides the expression of the enzyme DACS / DAOCS. Plasmid pPS52 is used to express the enzyme Cephalosporium either in Penicilliunio
The selected amds marker can be inserted into any expression vector. The pPS54 vector includes the amds gene from the plasmid pPS51 and the Penicillium IPS gene. Strains Penicill-ium possess some resistance to gidromitsin. Optimal transformation conditions for using HmR as a selectable marker require the addition of hydromycin-sensitive agents. The selection method for Penicillium transformants suggests transforming recombinant DNA containing the acetamide gene, Penicillium host cells, and cultivating the transformed cell in growth medium that contains acetamide as the only cell source of carbon or nitrogen.
The source of the acetamidase gene (amds) from Aspergillus nidulans can be the plasmid p3SR (NRELB-18182),
An intermediate vector was created, designated as plasmid pPS51, which consists of the amds gene from plasmid p3S2, fused with plasmid pMLC12. Plasmid pPS51 is digested with the HindIII restriction enzyme, treated with a Klenow fragment and stitched with a 2.13 kb fragment, previously with blunt ends, containing the hybrid gene DACS / / DAOCS, as a result of which plasmid pPS60 is obtained. Plasmid pPS60 contains the Penicillium IPS promoter, located adjacent, to express the DACS / DAOCS coding sequence. A deletion of two small restrictive Xbal fragments from the pRbBO plasmid creating the plasmid pPH58 is carried out. The synthesized binding fragment is inserted into Xbal; which ensures the operable binding of the promoter, pa Penici Ilium I IPS and the DACS / DAOCS coding sequence, resulting in the plasmid ppS59. Plasmid pPS59 is used to express the DACS / DAOCS activity in cells in Penicillium and other cells in which the Penicillium IPS promoter functions.
 Plasmid pPS59 is an intermediate for constructing expression vectors. Penicillium DACS / DAOCS
pPS 62 and pFS61. In both cases, x fragment 2.1 kb The bam HI-Nru I plasmid pPS59 with blunt-ended ends by Klenow enzyme is used as the source of the DACS / DAOCS coding sequence fused to the Penicillium IPS promoter. Plasmids pPS61 and pPS62 contain the selected markers on Penicillium ,. A fragment of 2.13 kb of plasmid pPS59 is cleaved with Hind III cleaved and the plasmid pPS57 prepared with Klenow fragment, and the plasmid pPS62 is obtained. Plasmid pPS62 is an expression vector.
The DACS / DAOCS promoter Cephalospori-um acremonium can also function in Penicillium (a discussion of the pPS52 plasmid is given), but the optimal Penicillium expression vectors use the promoter from the Penicilli gene. The Penicillium IPS promoter from plasmid pLCI (NKRLB-181 81) is bound to the skeleton of plasmid pMLC12 using a synthesized binding molecule, resulting in the production of plasmid ppS53. Plasmid pPS53 is an intermediate in the construction of some other cloning vectors and expression plasmids. The promoter-containing fragment of Bam, 0 kV plasmid pPS53 is fused to a fragment encoding a sequence, 3 tp0o. Bam HI of the plasmid pPS55, resulting in the formation of the plasmid pPS57.
Plasmid pPS57 is an intermediate in the construction of Penicillium pPS62 expression vector. The pPS62 plasmid contains a selectable marker (HmR) and a hybrid DACS / DAOCS gene with coding sequences for both DACS / DAOCS and HmR under the control of a promoter (2 copies) of the Penicillium gene
 Ips.
The expression vector Penicillium DACS / DAOCS without a selectable marker is constructed by inserting a 1.0 t „p fragment containing promoter. Bam HI plasmid pPS53 to the Bgl II point of the vector
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expressing pIT511 E, coli DNA encoding a DACS / DAOCS enzyme. Plasmid and derivatives that contain the target DACS / DAOCS gene are used to increase the production capacity of the Cephalosporium antibiotic in related cells of the recipient in which the C Jac promoter functions.
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The remonium and plasmid pPS56 translation activating sequence also includes the hydromycin resistance gene, which functions in C. acremonium and allows the selection of transformants in C. acremonium pPS56.
After selection of the transformer Cepha-losporium acremonium pPS56, there is no need to maintain the pressure of the selection of hydromycin B in the growth medium. Selective pressure is not required because the transformants C. acremonium pPS56 are very stable. This stability is the result of transformation by the plasmid pPS56, C o acremonium through chromosomal integration.
Transformed Penicillium cells can be selected using Hydromycin B0.
A method is proposed for the conversion of penicillin G and penicillin V to the corresponding cephalosporium using DACS / DAOCS in Penicillium, which involves the transformation of a host cell of the Penicillium with a recombinant DNA vector that contains a gene encoding the expression of the activity of DAOCSo
The plasmid includes genomic DNA of more than 3 kV, which is located below the DNA encoding DACS / DAOCS in the genome of Cephalosperium acremonium. Plasmid pIT503 includes a promoter and a sequence that activates the translation of the DACS / DAOCS gene
Plasmids pIT503, pPS52 and pPS56 include the promoter Cephalosporium acremonium and the sequence that activates the translation of the DACS gene / / DAOCSo -C.acremonium promoter and the sequence that activates the translation located on the plasmids PIT503, pPS52 and pPS56, can be used for a simulator. yes DNA sequences. The sequence of the promoter C. acremonium and the sequence that activates the translation is encoded on fragment 440 bp Sst I - Hind III, located directly above and in close proximity to the coding sequence DACS / DAOCS. The fragment that includes the specified restrictive fragment / v440 p. Sst I - Hind III, includes the promoter C. acremonium and the sequence that activates translation.
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The sequence of the promoter of Cershalosporium acremonium and the sequence that activates translation is encoded on a plasmid. Plasma mida contains a fragment of 440 p. Ssc I -. Hind III,
The promoter of Cephalosporium acremonium and the sequence that activates translation can be used to express a heterologous DNA sequence. The effect of the DACS / DAOCS promoter with a gene resistant to bacterial hygromycin B allows the use of selection using hydromycin B in Cephalosporium. Transcription termination sequence and other regulatory signals at the 3'-end of the DACS / DAOCS gene are described. Plasmid and its derivatives, such as plasmid / pPS56, additionally contain DNA of more than 1 kb. below the point of onset of transcription of the DACS / DAOCS gene in the genome of Cephalosporium acremonium.
A part of this Z-regulatory DNA sequence is known and described as follows.
W-TAS-W. is a translational terminal sequence:
 GGA ACC CGC CCA TOP AGT AAT AAA TCT ACG GGA GTT TAA GAA GAA AAA TTG CCC TAT AAA TTG CTA AAT TIT TAA AAC ACA AAG CAT GAG TGT CAA GAG TTT CAA GTT TCA A-3 This sequence and sequences that include 3-regu DACS / DAOCS gene sequence sequences can be introduced into recombinant DNA expression vectors
The cloning of a sequence that encodes a DAOCS enzyme was first performed. In addition, both the amino acid and DNA sequence of the expandase enzyme, especially DAOCS synthetase Cephalosporium acremo-, nium, can be used to separate the DNA encoding the expandand from the producing lactam organisms. This DNA sequence can be used to prepare labeled probes, intended for the detection of expandase-encoding DNA sequences in lactam-producing organisms. The content of G and C in DAOCS coding DNA, which is approximately 63%, leads to the fact that these DNAs are suitable for the isolation of Sfcreptomyces DNA coding for DAOCS, mainly S. clavaligerus. DNA
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Streptomyces has a high content of G and C and reaches 70%. Such a high content of G and C in DNA allows them to be used to isolate homologous S.slavaligerus or other DNA sequences encoding DAOCS.
Example 1. Culture E „coH K12 JA 221 / p1T508 and isolation of plasmid.
1 l of nutrient broth L (10 p of tryptone, 10 g of NaCl and 5 g of yeast extract per 1 l) containing 50 μg / ml of ampicillin is inoculated with E. coli K12 JA 221 / pIT503 and incubated in an incubator at 37 ° C until until optical density at 590 nm is 1 unit. absorption. During this time, 150 mg of chloramphenicol is introduced into the culture. The incubation is continued for 16 hours. The introduction of chloramphenicol inhibits the synthesis of protein l, thus inhibiting further cell division, but provides further replication of the plasmid.
This culture was centrifuged at a rotation speed of 6000 rpm for 5 minutes at 4 ° C. The resulting surface layer was removed and the cell granule was washed in 40 ml of TES buffer solution (10 mM Tris-HCl; pH 7.5; 10 mM NaCl and 1 mM ethylenediaminetetraacetic acid (EDTA) are then granulated again. The surface layer is removed and the cell granule is frozen in a dry ice-ethanol mixture tank and then thawed. The deflated cell granule is suspended again in 10 ml of solution 25% sucrose and 50 mM EDTA. Into this solution are injected and mixed with it 1 ml of l zo- tsimnogo solution concentration of 5 mg / ml 3 ml 0, 25 M EDTA, pH 8.0 and 100 .mu.l of ribonuclease A concentration of 10 mg / ml The mixture was then incubated on ice for 15 min..
3 ml of lysing solution (prepared by mixing 3 ml of 10% Triton-X 100.75 ml of 0.25 M EDTA, pH 8.0, 15 ml 1 M Tris-HCl, pH 8.0 and 7 ml of water) is stirred and the resulting solution is incubated on ice for another 15 minutes. Lysed cells are frozen in a dry ice-ethanol bath and then thawed.
Cell debris is removed from the solution by centrifuging during rotation of the centrifuge at a speed of 25,000 rpm for 40 minutes. The surface layer is extracted with buffer phenol. 30.44 CsCl and 1 ml of ethylbromide solution with a concentration of 5 mg / ml are introduced into this solution. Then i, this solution is brought to a volume of 40 ml and decanted into an ultracentrifugal tube. This tube is hermetically sealed and the solution is centrifuged in a rotary centrifuge at a centrifuge speed of 42,000 rpm for 16 hours. The resulting plasmid, visualized with ultraviolet light, is isolated and then placed in a tube and a rotary centrifuge, where centrifugation is carried out1 at a rate of 50,000 rpm within 16 hours. Any necessary volume control is carried out using the TEZ solution containing 0.761 mg / ml CsCl. The plasmid band was then isolated, extracted with saturated salt isopropanol to remove ethyl bromide, and diluted (1: 3) with TEZ buffer solution. Two volumes of ethanol were added to the solution and then it was incubated at -20 ° C overnight. Plasmid DNA is granulated by centrifuging this solution in a rotary centrifuge for 15 minutes at a centrifuge speed of 10,000 rpm.
Plasmid pTT3T DNA obtained in this way (1 mg) is suspended in 1 ml of TES buffer solution (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and stored at -20 ° C.
The sequences of the terminal codon of the 1pp coding sequence are replaced by Bam HI by introducing the synthesized binding DNA molecule (5 -CCGGATCCGG-3). The coding sequence of the last thirty five amino acids of the lipoprotein, the translational terminal signal and the sequence corresponding to the 3-untranslated region of the informational RNA, follows the Wat H1 point. The pKE N021 plasmid also includes some independent 850t0ooo sequences located below the 1рр gene in the chromosome E.CON.
Approximately 50 μg of plasmid pKENIII are treated with 25 Hpal restrictases in 300 μl of Nr IX buffer
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(10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM KCl; and 6 mM | 3-mercaptoethanol) at 37 ° C for 2 hours. The mixture is extracted twice with 300 ml of a mixture of phenol and chloroform in a ratio of 50: 50 and the extracted aqueous phase is re-precipitated 2.5 vol. ethanol and 0.1 about 3 M NaOAc. The DNA bead is dissolved in 100 µl of electrophoresis buffer solution and fractionated on a 5% polyacrylamide gel.
PRI mme R 2. Construction of the plasmid RGH.
The construction of plasmid pKEN021 and the selection of a fragment of 5.1, etc. „about. Xba I - Bam HI is carried out as follows.
Plasmid pKEN021 is obtained from plasmid 0 pKENIIIo. Plasmid pKENIII is obtained from the Northern Regional Research Center (NRRL), the Agricultural Research Service, Perori, Illinois, 61604, EoColi CC620, the order number NRRL 1501 К
Plasmid pKENIII has a fragment of l-2.8 to EcoRI, which includes the gene E.coli 1pp.
In the plasmid pKEN021, the 650 bp EcoRI - Sal I fragment of the plasmid pBR322 is replaced with the gene sequence of E. coli 1pp. These 1pp gene sequences include a fragment of 462 n „o. Alu I, located upstream of the first triplet of the 1pro coding sequence. This fragment of l / 462 Lp contains the promoter of the 5-untranslated zone and the point of ribosomal binding. 0 The unique Xbal restriction site is located within the ribosomal binding point 16 then in front of the methionine initiating translation. The Pvu restriction site, located above bis, is 29: 1 in all polyacrylamide gels, except in special cases. This gel is stained in a solution containing 0.5 μg / ml ethyl bromide, and a DNA band is visualized in long-wave ultraviolet light. Fragment 950 p. Hpall is isolated and removed from the gel by electrolysis into a dialysis bag. After extraction with phenol / SSCS and precipitation with ethanol, DNA (2.5 µg) was dissolved in 25 µl of TEN (10 mM NaCl; 10 mM Tris-HCl, pH 7.4, 1 mM EDTA).
Approximately 2 μg of fragment 950 p. .Hpall is treated with Alu I enzyme
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in 200 μl of IXAlu I solution (50 mM NaCl, 6 mM Tris-HCl, pH 7.6, 6 mM MgCl and 6 mM / 3-mercapto ethanol) for 2 hours at 37 ° C. DNA is fractionated in 6% polyacrypamide gel, fragment 462 Po o. Alu I is extracted, (A / I µg). Dissolved in 10 ml of a solution (66 mM Tris-HCl, pH 7.6, 10 mM MgCl ,, 10 mM dithiothreitol, 0.4 mM ATP) containing 150 pmol of phosphorylated EcoRI binding unit (p -CCAATTSS-3) and 2 units. T4 DNA League. After incubation at 4 ° C for 16 h, the mixture is heated at 65 ° C for 10 min and diluted to a solution composition: 100 mM Tris-HCl, pH 7.2, 50 mM NaCl, 10 mM 6 mM / 3-mer - captoethanol containing 40 units of EcoRI enzyme. After 2 hours at 37 ° C., the sample is extracted with a phenol / CHC1 mixture and precipitated with ethanol. The DNA is then dissolved in 20 µl of a T4 DNA ligase buffer solution containing T.4 DNA ligase and 0.1 µg of alkaline phosphatase treated and EcoRI enzyme plasmid pBR3220. After crosslinking at 4 C for 16 h, DNA is used to transform E. coli K12 HB101 (NRRLB-15626). Transformants are selected on agar plates containing 12 μg / ml of tetracycline and plasmids isolated from persistent colonies by rapid alkaline extraction. Plasmid containing a fragment of 466 bp. Xba I - Bam HI, used as starting material for the next step.
Approximately 2 μg of this plasmid, treated with 2 units. Hind III enzyme in 50 μl of a buffer solution IX Hind III (60 mM NaCl; 10 mM Tris-HCl, pH 7.4, 10 mM MgClgH 6 mMr-mercaptoethanol) for 1 h at 37 ° C, extracted with a mixture of phenol / SSL is precipitated with ethanol, dissolved in 200 µl of nuclease IX SI buffer solution (300 mM NaCl; 30 mM NaOAc, pH 4.25, 3 mM ZnCLj) and treated with 200 units. iuclease S1 for 1 h at 15 ° C. The reaction is terminated by extraction with phenol / CHCl3; the DNA is precipitated with ethanol. It is treated with Hind III enzyme, dissolved in 10 ml of T4 DNA ligase buffer solution containing 20 pmol of phosphorylated Bam HI binding units (S -CCGGATCCGG-S) and 2 units. T4 DNA ligase. After 16 h of incubation at
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65 ° C for 10 minutes and then diluted to 100 ml to obtain a IX Bam HI buffer solution (150 m NaCl; 20 mM Tris-HCl, pH 8.0, 10 6 mM / $ - mercaptoethanol) containing 20 items the enzyme Wat H1 “After 2 hours of incubation at 37 ° C, the mixture is purified on a 1% agar gel. This gel is stained with ethyl bromide, and a larger fraction (4.5 kb) is recovered from g l.
The fragment has cohesively bonded ends of Bam HI. It is dissolved with 20 µl of T4 DNA ligase buffer solution containing T4 DNA ligase. After 16 h of aging at 4 ° C DNA and used for transformation of E.sub.1g K12 HB101 (NRRLB-15626) Transforms are selected for resistance to ampicillin at 100 mg / ml and sensitivity to tetracycline at 10 m / ml. Plasmids of selected colonies are followed by the absence of the Hind point and the presence of a single point. Wat H1 Sequential hydrolysis with EcoRI, Sail results in the formation of two 46b and 3x bp fragments. and a significantly longer fragment. A plasmid with such characteristics is modified to transform the EcoRI point located above the 1pp promoter into the restrictive Hind III point.
The modification is carried out by partial hydrolysis with 2 µl of plasmid 100 µl of Eco Buffer Solution IX with restrictive enzyme EcoRI. The reaction is stopped by heating to approx 40,651. The DNA is extracted with a fen / CHC1 mixture and precipitated with ethanol. The DNA is digested in a 200 ml Nuclease IX S1 buffer solution containing 1000 / ml S1 nuclease and incubated with
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45 120 ° C for 1 hour. The reaction is stopped by extraction with phenol / CHC DNA and precipitated with ethanol and suspended in 10 μl of T4 DN ligase buffer solution containing 20 pmol phosphorus
50 hinyi III binding agent (5 -CCAAGCTTGG-3 H 2 units of T4 D ligase. After 16 hours of incubation, the mixture is heated for 10 minutes at 65 ° C, diluted to 150 µl with a half
55 according to the composition of the buffer IX Hind I | containing 10 units. restrictive enzyme Hind III, incubated for 2 h at 37 ° C and then fractionated
.4 The reaction mixture is heated at -h. Ruta in 1% agarose gel. DNA
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65 ° C for 10 min and then diluted to 100 ml to obtain a IX Bam HI buffer solution (150 mM NaCl; 20 mM Tris-HCl, pH 8.0, 10 mM 6 mM / $ - mercaptoethanol) containing 20 units . enzyme Wat H1 “After 2 hours of incubation at 37 ° C, the mixture is purified on a 1% agarose gel. This gel is stained with ethyl bromide, and a larger fragment (4.5 kb) is recovered from the gel.
The fragment has cohesively bonded ends of Bam HI. It is dissolved in 20 µl of T4 DNA ligase buffer solution containing T4 DNA ligase. After 16 hours of incubation at 4 ° C, the DNA is used to transform the E. coli K12 HB101 (NRRLB-15626). Transformants are selected for ampicillin resistance at 100 mg / ml and for tetracycline sensitivity at 10 μg / ml. Plasmids of the selected colonies are examined for the absence of the Hind III point and for the presence of a single point of Wat H1 Sequential hydrolysis of EcoRI, Sail leads to the formation of two fragments of 46b and 3X15 bp. and a significantly longer fragment. A plasmid with such characteristics is modified to convert an EcoRI point located above the 1pp promoter to the Hind III restriction point.
The modification was carried out by partial hydrolysis of 2 µl of the plasmid in 100 µl of EcoRI buffer IX with the restrictive enzyme EcoRI. The reaction is stopped by heating at 40,651. The DNA is extracted with a phenol / / CHC1 mixture and precipitated with ethanol. The DNA is dissolved in 200 ml of nuclease IX S1 buffer solution containing 1000 units / ml nucase S1, and incubated with
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45–20 ° C for 1 h. The reaction is stopped by extraction with phenol / CHCl – jo DNA and precipitated with ethanol and suspended in 10 μl of T4 DNA ligase buffer solution containing 20 pmol of phosphorus Hind III binder (5 -CCAAGCTTGG-3 H 2 units T4 DNA ligases. After 16 h incubation at 4 ° C, the mixture is heated for 10 minutes at 65 ° C, diluted to 150 µl with the preparation of the composition of buffer IX Hind III containing 10 units of the restrictive enzyme Hind III, incubated for 2 h at 37 ° C and then fractionation23;
recovered, purified, dissolved in 20 µl of T4 ligase buffer solution containing T4 ligau, incubated for 16 h at 4 ° C and used to transform HB101 (NRRL B-15626). The plasmid DNA transformant ApR analyzed, Select a plasmid with a fragment of 500 p. EcoRI - Hind III. This plasmid is then used as a cloning vector for the 3-zone of the 1pp gene. 2 μg of this plasmid is hydrolyzed in 50 μl of Sal I IX buffer solution (150 mM NaCl, 6 mM Tris-HC1 pH 7.9, 6 mM MgCl2H 6 mM / 3-mercapto-etanrla) with 2 units. Sal I enzyme for 1 h at 37 ° C. The reaction mixture was diluted to 150 µl to obtain a BamHI buffer solution composition containing 2 units. the enzyme bam hi. After incubation for 1 hour at 37 ° C, about 2.5 units are added. alkaline phosphatase and incubated at 65 ° C for 1 h. The DNA is extracted with a phenol / CHCl 3 mixture, precipitated with ethanol, dissolved in TEN and used as a cloning vector for the 3 -1 pp fragment.
To obtain a 1p3 fragment, 10 µg of the plasmid pKENIII is hydrolyzed in 200 µl of IX Hpal buffer solution containing 10 units. Hpal enzyme, for 2 hours at 37 ° C. After extraction with phenol / CHC and precipitation with ethanol, the DNA is dissolved in 10 µl of T4 ligase buffer DNA containing 20 pmol of Sal I phosphorylated binder (5 -GGTGACC-37 ) and T4 DNA ligase. Incubate for 16 hours at 4 ° C. Inhibit ligase by heating at 63 ° C for 10 minutes. The resulting material is diluted to a volume of 100 µl to obtain a composition composition of a IX Sal I buffer solution containing 10 units. enzyme Sal I, and incubated for 1 hour at 37 ° C. The reaction mixture is diluted-up to 300 µl, get; the composition of the buffer solution IX Pvu II (60 mm NaCl, 6 mm Tris-HCl, pH 7.5, 6 mm 6 mm uZ-mercaptoethanol) containing 10 units. Pvu II enzyme. After 1 hour of incubation at 37 ° C, DNA is fractionated on a 5% polyacrylamide gel. 0.5 µg of fragment 950 bp. Is extracted, purified and dissolved in TEN.
0.2 mg of this fragment is diluted in 20 µl of T4 DNA ligament buffer solution containing 20 pmol phosphorus
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Bam HI linking link (5 -CCGGATCCGG-37) and 2 units. T4 DNA ligases, and incubated for 16 h at 4 ° C. The obtained DNA is heated for 10 minutes at 65 ° C, diluted to a volume of 100 µl to form a IX Bam HI buffer solution composition containing 20 units. enzyme Bam HI, incubated at 37flC for 2 hours and then fractionated in 5% polyacrylamide gel to remove excess binding molecules
The resulting fragment 950 p. with cohesively bound ends of Bam HI and Sal I are purified and dissolved in 20 µl of T4 DNA ligase buffer solution containing 0.2 µg of Bam HI - Sal I hydrolyzed plasmid 105 and T4 DNA ligase. After incubation for 16 h at 4 C DNA is used to transform E, coli K12 HB101 (NRRL B-15626) p Plasmid clones are analyzed for the presence of the Sal I fragment — 25 Bam HI, and the plasmid (5.2 tons bp) is selected, which is designated pKEN021 .
10 mg of plasmid pKEN021 is hydrolyzed in 200 ml of Bam HI buffer solution containing 10 units. each of the Bam Hi and XBa I enzymes for 1 hour at 37 Cs. Then 2.5 units of alkaline phosphatase are treated for 1.5 hours at 65 ° C, extracted with a mixture of phenol / CHS and precipitated with ethanol and dissolved in 50 ml of TEN for subsequent use
Construction of the plasmid pNM575 is carried out as follows.
The pt-EDSOch GH 800 plasmid is used as a source of DNA containing parts of the hGH coding sequence that contains a unique restriction point 6 pt. Oo below the translational codon of this coding sequence. This point is replaced with a Bam HI point by the method described. 6 μg of the ptzpEDSOch GH 800 plasmid is hydrolyzed with 6 g of Sma I in 200 ml of solution IX of Sma I (15 mM Tris-HCl, pH 8.0, 6 mM MgCl2, 15 mM KC1 and 6 mMUE-mer-capto-ethanol) 15 hours at 37 ° C, extracted with phenol / SSCC, precipitated with ethanol and dissolved in 24 µl of TEN containing 40 pmol of Vayi phosphorus binding link. The mixture is incubated for 2 hours at 4 ° C. ° C, and then for 10 min at 65 ° C.
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Cohesively linked Bam HI terminal units are formed by hydrolysis with the restrictive enzyme Bam HI. Hydrolysis leads to the formation of a fragment of 691 bp. with cohesively connected ends of Bam HI.
The DNA fragment was stitched with 0.2 μg of hydrolyzed Bam HI and alkaline phosphatase-treated DNA plasmid pBR322o. After 16 hours of incubation at 4 ° C, this material was used to transform E. coli K12 JA 221 (NRRL B-15014). Transformants were selected on agar plates. containing 100 µg / ml ampicillinao Plasmids are isolated and identified by restriction enzyme analysis and gel electrophoresis method. The desired plasmids, designated pNM575, contain a fragment of 700 p. Bam HI.
Construction of plasmids RI are as follows.
The coding sequence of mature hGH contains one Fnu PII point of 47 bp. from the first nucleotide translational
5 -CTAGAGGGTATTACATATGGATTTCCCAACCATTCCCCTCTCGAGGCTTTTTGACAACGCTATGCTCCG-3
And I I t I II l | l I 1 1 II And I And I M I I I And I I And I P M II And I And I I I I I I 111 1 And P I U | t 3 -TCCCATAATGTATACCTAAAGGGT (tGGTAAGGGGAGAGCTCCGAAAAACTGTTGCGATACGAGGC-5)
This fragment is obtained by the phosphoriferine method, by which the following fragments are prepared:
1) 5 -CTAGAGGGTA-3;
2) 5 -TTACATATGGATTTCC-3;
3) 5 -CAACCATTCCCCTCTCGAGGC-3;
4) 5 -TTTTTGACAACG-3;
5) 5 -CTATGCTCCG-3;
6) 5 -CGGAGCATAGCGTT-3;
7) 5 -GTCAAAAACCCT-3;
8) 5 -CGAGAGGGGAAT-3;
9) 5 -GGTTGGCAAATC-3 ;;
10) L -CATATGTAATACCCT-3. Using them, a stepwise reaction of the catalyzed binding of T4 ligase is carried out:
b - non-phosphorylated segment 1 is mixed with phosphorylated segments 2 ,. 9 and 10 and exposed to T4 ligase with the formation of duplex 1 DNA; this duplex is extracted by the method of preparative gel-elactrophoresis in 15% polyacryls de;
5-unphosphorylated segment 6 is mixed with phosphorylated segments 3, 4, 5, 7 and 8 and exposed to T4 ligase DNA to form a duplex form of DNA 2; this
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initiating point. 25 μg of the plasmid rsH575 are hydrolyzed in 250 μl of the Bam HI solution with 25 units of the Bam HI enzyme at 37 ° C for 1 hour. Fragment 691 bp. Bam HI is extracted from a 6% polyacryl anide gel and one third of this fragment is purified (equivalent to 8 μg of plasmid) is hydrolyzed in 100 μl of Fnu PII solution (6 mM NaCl; 6 mM Tris-HCl, pH 7.4, 6 mM MgCl and 6 mM | 3-mercaptoethanol) with 2.5 units. the Fnu PII enzyme for 1.5 hours at 37 ° C. Electrophoresis in a 6% polyacrylamide gel and standard extraction procedures are used to extract a 538 bp DNA fragment containing the coding sequence of the last 175 amino acids of hGH, followed by the translation of the stop signal.
A double-stranded DNA fragment is synthesized by an eusphothreether method, connecting the 1pp promoter zone with the hGH coding zone. This double-stranded DNA fragment has the following sequence:
duplex purified by electrophoresis in a 15% polyacrylamide gel;
DNA duplexes 1 and 2 are joined together by T4 ligase, forming the DNA
duplex 1100, which is purified by electrophoresis in a 10% polyacrylamide gel; This DNA duplex is enzymatically phosphorylated using T4-polynucleotide kinase and Ј-32 P-ATP, using the procedure described.
The plasmid is constructed by linking 0.1 pmol (0.4 µg) of the Xba I-Bam HI fragment of the plasmid pKEN021, 0.025 pmol of the synthesized DNA fragment and 0.3 pmol (0.08 µg) of the fragment 538 p „o„ plasmid pNM575 in 24 µl solution using T4-DNA DNA. After incubation for 1 h at 4 ° C, the mixture is transformed with E. coli JA 221 (NRRL B-15014) Transformants are selected on agar plates containing 110 μg / ml ampicillin and cultured.
Construction of plasmid rIOS lead
in the following way.
The hGH gene sequence in the plasmid contains the Xho I point on 24 bases below the translational initiating point and the vXba I point in
non-coding zone 5. 25 μg of the plasmid is hydrolyzed in 250 μl of the Bam HI solution with 25 units. Cha I and 25 units. Xho I at 37 ° C for 1 h. A large fragment is purified from agarose gel according to the described method,
Double-stranded DNA fragment is synthesized by the phosphotriester method to input short open reading frame (cistron) in front of kodi5 -CTAGAGGGTATTAATAATGTATATTGATTTTAATAAGGAGGAATAATCATATGGATTTCCCA a -TCCCATAATTATTACATATAACTAAAATTATTCCCTCCTTAGTATACCTAAAGGGT
1) 5 -CTAGAGGGTATTAATAATGCATATTGATTTTAATAAGGAG-3;
2) 5 -GAATAATCATATGGATTTCCCAACCATTCCCCTC-3;
3) 5 -TCGAGAGGGGAATGGTTGGGAAATCCATATGATTATTCCTCCTT-3
4) 5. -ATTAAAATSAATATATSATTATTAATASSST-3
Using the prepared segments, the compound is made by mixing 5 - non-phosphorylated segments 1 and 3 with 5-phosphorylated i segments 2 and 4 and exposing the mixture to a T4 ligase. The resulting duplex is purified by electrophoresis in a 10% polyacrylamide gel. The duplex DNA is then enzymatically phosphorylated using a T4 polynucleotide mixture and incubated at 16 ° C for 1 hour, the reaction is completed by extraction with phenol and the DNA is purified. The hydrolyzed Nde I and the Klenow fragment treated with the Klenow fragment were crosslinked with T4 ligase 25 DNA at 4 ° C for 16 hours. The resulting DNA was used to transform the RV308 strain of E. coli K12 (NRKL B-15624). Transformants are selected on L-agar plates containing
. ... ™ .L ™ a3 ZO Juo µg / ml ampicillin Plasma grade LGH pPOS is constructed by stitching 0.1 pmol (0.4 µg) of the Xba I - Xho I fragment of the plasmid with 0.025 pmol of the synthesized DNA fragment in 24 µl of solution, s using T4 ligase DNA. After incubation for 1 h at 4 ° C, the mixture is used to transform E. coli JA 221 (NRRL B-1S014). Transformants are selected on agar plates containing 100 μg / ml ampicillin and cultured in the usual manner.
The construction of E. coli K1 2 RV 308 / / pL 110 A is carried out as follows.
1 μg of plasmid pL110 DNA is hydrolyzed 3 units. Nde I enzyme in a volume of 20 μl of a solution of 1.0 M NaCl, 0.50 M Tris-HCl, pH 7.5, 0.10 mM MgCl2 and 10 mM dithiothrityol for 1 h at 37 ° C, extracted with phenol / chloroform and precipitated with ethanol. The plasmid pLIlO DNA was dissolved in 50 µl of Klenow IX solution (40 mM KROi, pH 7.5, 6.6 mM MgCl2, 1.0 mM 2-mercaptoethanol, 33 µM d-ATP 33 µM d CTP; 33 µM d -GTP and 33 μM TTP). Two μl (10 units) of a large DNA fragment from E. coli gulymerase 1, known as the Klenow fragment, is introduced into this DNA and mixed with it. Reactionary
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divided from the resulting colonies by rapid alkaline extraction. So select plamidu ryua not containing the point Nde It
The construction of the pLMOB phage by point-specific mutagenesis is carried out as follows.
To construct M13Ts3 phage, 50 µg of the plasmid RYUV in 50 µl of TEN solution was introduced into 25 µl of 10X Hind III solution and 170 µl of water, 5 µl (V50 food) of Hind III was added and incubated at 37 ° C for 2 hours. 13 μl of 2 M Tris-HCl, pH 7.4 and 5 μl (50 units) of the EcoRI enzyme are incubated and incubated for more than 2 hours at 37 ° C. The reaction is stopped by extraction
phenol, saturated TEN, phenol is removed by extraction with chloroform. Plasmid DNA was precipitated, centrifuged, electrophoresed on a 1% agarose gel, and the EcoRI - Hind III fragment of 4.3 kb was isolated.
Approximately 5 µg of m13mp18 55 phage DNA was dissolved in 50 µl of TE solution, hydrolyzed with Hind III and EcoRI enzymes as described, Hind III-EcoR cut, and a fragment of 25 T.O. was purified.
hGH sequence. This double-stranded DNA fragment has the indicated sequence:
ASSATTSSSSTS-3 TGGTAAGGGGAGAGCT-5
(ABOUT
This fragment is obtained by the method of recognition phosphotriester method, through which receive the following segments:
the mixture is incubated at 16 ° C for 1 h, the reaction is completed by extraction with phenol and the DNA is purified. The hydrolyzed Nde I and the Klenow fragment treated with the Klenow fragment are stitched with T4 ligase DNA at 4 ° C for 16 hours. The obtained DNA is used to transform the RV308 strain of E. coli K12 (NRKL B-15624). Transformants are selected on L-agar plates containing
Yuo µg / ml ampicillinPlasma from ды5
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divided from the resulting colonies by rapid alkaline extraction. So select plamidu ryua not containing the point Nde It
The construction of the pLMOB phage by point-specific mutagenesis is carried out as follows.
To construct M13Ts3 phage, 50 µg of the plasmid RYUV in 50 µl of TEN solution was introduced into 25 µl of 10X Hind III solution and 170 µl of water, 5 µl (V50 food) of Hind III was added and incubated at 37 ° C for 2 hours. 13 μl of 2 M Tris-HCl, pH 7.4 and 5 μl (50 units) of the EcoRI enzyme are incubated and incubated for more than 2 hours at 37 ° C. The reaction is stopped by extraction
phenol, saturated TEN, phenol is removed by extraction with chloroform. Plasmid DNA was precipitated, centrifuged, electrophoresis was performed on a 1% agarose gel, and the EcoRI – Hind III fragment was extracted with 4.3 kb.
Approximately 5 µg of m13mp18 5 phage DNA was dissolved in 50 µl of TE solution, hydrolyzed with Hind III and EcoRI enzymes as described, cut off with Hind III-EcoRI, and a fragment of 25 T.O.
29
100 ng of the fragment, 3 t0p, o. Hind III - EcoRI plasmids RSA is mixed with 100 ng of fragment / 7.25, etc., o. Hind III - EcoRI phage M13 mp18, 2 μl of ligase 10X buffer solution,
1 μl units „) T4 ligase DNA and 1A μl. The mixture is incubated at 15 ° C for 1.5 hours.
1 ml. night culture of E. coli K12 jM 109 E. coli K12 jM 101 is used to inoculate 50 ml of nutrient broth L, the resulting culture is incubated at 37 ° C with aeration until ODgg0 reaches a value from 0.3 to 0 ,four. The cells were resuspended in 25 ml of 10 mM NaCl, incubated on ice for 10 minutes, collected by centrifugation, resuspended in 1.25 ml of 75 mM CaClg, an aliquot (200 µl) of these cells was removed, introduced into 10 µl of cross-linked DNA, cooked as indicated and incubated on ice for 40 min. Then incubated at 42 ° C for
2 min, aliquots (1, 10 and 100 µl) are removed and injected into 3 ml of upper agar (L nutrient broth with 0.5% agar maintained in the melted state at 45 ° C), which also contains 50 μl of 2% X-Gal, 50 μl of 100 mM IPTG and 200 μm of E. coli K12 jM 109 c. logarithmic growth phase). The cell mixture - upper agar is placed on plates with L agar containing 40 μg / ml X-Gal (5-bromo-4-chloro-37Indolyl- | 5-B-thiogalactoside)
and 0.1 mM IPTG (isopropyl-B-B-thiogalactoside). Plates are incubated at 37 ° C. overnight.
Transparent colonies are used to inoculate 2 ml of nutrient broth L, the resulting cultures are incubated at 37 ° C with aeration for 2 hours (the absence of blue color indicates that the desired DNA insertion has occurred). Then culture. centrifuged and 200 µl of the resulting surface layer was introduced into 10 ml of culture (0oD, S5D 0.5). E. coli K12 jM 109 grown at 37 ° C with aeration. These cultures are incubated for an additional 30 minutes at 37 ° C, and then the cells are granulated by centrifugation and used to obtain the replicative form of the recombinant phage that they contain. A double replicative form of phage DNA was isolated from cells using the procedure described in Example 1.
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Transformants containing the DNA of PHIZT3 phage are identified by analyzing the restrictive enzyme of their phage DNA o
5 To obtain single-stranded DNA of phage t13TsZ, 1.5 ml of a night culture of E. coli K12 jM 109 / m13Tc3 is subjected to centrifugation and 100 μl of the surface layer containing the phage t13TsZ is used to inoculate 25 ml of a culture of E. coli jM 109 at O.P. .6bo 0.4-0.5. The culture is inoculated for 6 hours at 37 ° C with aeration. During this time, the culture is centrifuged and the resulting surface layer (20 ml) is transferred to a new tube. 2 ml of solution containing 20% polyethylene glycol (PEG) 6000 and 14.6%
2Q NaCl, introduced into this surface
centrifugation layer, which is then incubated on ice for 20 minutes. The resulting surface layer is centrifuged for 25 minutes. The precipitate is again suspended in 500 μl of TE buffer solution. The DNA solution is extracted twice with saturated phenol TE and twice with chloroform. Single-stranded DNA is then precipitated using NaOAC and ethanol, and centrifuged. The precipitate is washed with 70% ethanol, dried and dissolved in 60 µl of H20.
Mutagenesis is carried out as follows.
35 A single-stranded DNA fragment used in mutagenesis is synthesized in an automated DNA synthesizer. This fragment has the specified sequence:
40 5 -CCCGTCCTGIGGATACTCTACGCCGA-3 It is homologous to the zone surrounding the Bam HI point (57-GGATCC-3}) in the tetracycline resistance gene of the plasmid pBR322, with the difference that the group 45 pa A, second from the 5 end (or one third of the 31 end), is C in the plasmid pBK322. Such a substitution does not alter the amino acid by bringing tetracycline resistance to protein, but eliminates the Bam HI point.
Approximately 10 pM of mutant seed and universal seed M13 are treated separately with 10 units. (BRL) T4-55 dinucleotide oxidase in 20 μl of kinase IX solution (60 mM Trie-HCl, pH 7.8, 15 mM 2-mercaptoethanol, 10 mM MgCl. | And 0.41 μM w-ATP) for 30 min at 37 C. Treated with kinase DNA
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used in the operation of mutagenesis, for which 300 ng 0.2 μl of monofilament phage t13TsZ, 1 pM (2 μl) of universal seed, 1 pM (2 μl) of mutagenic seed, 2 μl of 10 × analytical buffer solution (100 mM Tris-HCl, pH 7.5, 1 mM EDTA and 500 mM NaCl), and 12.8 μl of H2O are incubated at 80 ° C for 2 minutes, at 50 ° C for 5 minutes and cooled to room temperature.
5 µl of 10X solution (500 mM Tris-HCl, pH 8, 1 mM EDTA. 120 mM MgCl4), 5 µl of 2 mM d ATP, 1 µl of 6 mM solution in each of the TTP and d CTP, 1 µl (unit) of the enzyme Klenow, 1 μl (100 units) of T4 ligase DNA and 17 μl of ana- lyzed DNA are incubated at room temperature for 1 hour, at 37 ° C for 2.5 hours and then overnight at 4 ° C.
The reaction is stopped by two extractions with saturated TE with phenol and two extractions of DNA are precipitated with ethanol and NaOAc, precipitated by centrifugation, re-suspended in 50 µl of HgO and 6 µl of 10X 1 buffer solution is added to the DNA solution.
The DNA solution is distributed equally in the three tubes. Approximately 200 units are introduced into two of them. SI nuclease One reaction mixture is incubated at room temperature for 5 minutes and the other for 10 minutes. The reaction is stopped by extraction of the reaction mixture with saturated TE with phenol, extraction with chloroform and precipitation with ethanol. A non-processed DNA sample serves as a negative control sample.
The DNA pellet is re-suspended in 20 μl. 10 μl of the resulting solution was used to transform E. coli.
The double-stranded replicative form of DNA was isolated from about 48 colonies as described and selected for the presence of a Bam HI point (pLUOB). Isolates without a Bam HI point are selected as described by single-stranded DNA.
To construct the plasmid RYUS 50 µg of the replicative form of the DNA of phage RYUV hydrolysis, comfort in 250 µl of IX Nhe I buffer solution (50 mM NaCl, 6 mM Tris-HCl, pH 7.5, 6 mM MgC12 and 6 mM B-mercaptoethanol) containing l / 50 units Nhe I enzyme at 37 ° C for 2 hours. Add 5 µl of 5 M NaCl,
,

, |
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and then 5 μl (50 units) of the Sal I enzyme and hydrolyzed for 2 hours at 37 ° C. Fragment 422 p. Nhe I — Sal I containing the zone imparting resistance to tetracycline was isolated from an acrylamide gel,
The DNAs of the PKOXA plasmid are hydrolyzed with the enzymes Nhe I - Sal I under the same conditions, to obtain the Nhe I - Sal I fragment of 6.1 t „na.
100 ng of each of the obtained fragments are stitched using the usual Plasmid pL110C procedure with -. c gives E.coli resistance to tetracycline, but does not have a Bam HI point in the tetracycline gene.
To construct plasmid pCZRIII, fragment / 3.5 kbp is crosslinked. EcoRI - Ps I plasmid pL110 C (containing the intact coding sequence) with a fragment of 3 t.Poo. EcoRI - Pst I plasmid pIT160. The plasmid contains a fragment of 3 kb. EcoRI - Pst I, derived from the plasmid RYUUS inserted into the hydrolyzed EcoR I - I plasmid PUC18. The fragment contains the tetracycline resistance gene with the difference that point C1a I is excluded. The plasmid carries resistance to 10 µg / ml of tetracycline and there is no restrictive point C1a I. 50 µg of plasmid DNA pL110C is incubated in a volume of 250 µl of the IX EcoRI solution containing 10 µl (units,) EcoRI, for 2 h at 37 ° C. Then, 1 µl (5 units of) Rst I enzyme is introduced and the incubation is continued at 37 ° C. Aliquots are removed approximately every 5 minutes and the phenol / CHC mixture is extracted. These aliquots are collected and the mixture is subjected to agarose gel electrophoresis. Desired EcoRI - Pst I fragment A / 3.5 t „p. O. contains the un exposed gene bGII and includes the internal point Pst I. The fragment is cleaned and stitched with a fragment of Pst I - EcoRI of 3 kb. plasmids rGPbO. The obtained 50 DNA transform E. coli K12 jM 109 and the desired plasmid is designated pCZRIII, characterized using known methods.
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To construct the plasmid pCZR336, 50 µg of the pIO3 plasmid are incubated in 250 µl of high salt buffer solution IX containing half 50 units. the enzyme bam hi and cha i for
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2 h at 37 ° C. Fragment 650 p. Of XA I-Bam UI, containing the first cisterone and the LGH coding sequence, is separated and purified on an acrylamide gel. 50 μg of the plasmid pCZBIII is hydrolyzed with Xba I and Bam HI enzymes and a large fragment is purified. The fragments are stitched together and transformed into E. coli K12 RV308, the plasmid pCZR33S is identified. Construction of the plasmid.
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b -TATGACTTCCAAGCTTCCCGTCTTTCGTCTAGACGACCTCAAGAGCGGCAAGGTCCTCACCGAGCT-S
I I I M I I I III I I I I I I I I I III III I I I I I I I III III I AC 1 I I HI H I AND I 3 -ACTGAAGGTTCCAAGGGCAGAAAGCAGATCTGCTGGAGTTCTCGCCGTTCCAGGAGTGGC-5
This binding molecule was synthesized in a DIC automatic synthesizer in the form of two single strands. The molecule has several changes; the formation of a point Nde I including a translational initiating point; replacing C with A in the third position of codon 10 (leucine codon), since CTC and CTA are equivalent, forming the point Xa I; replacing C with T in the third position of the fourth codon (valine codon), since GTC and GTT are equivalent.
5 µg of pUC 19 plasmid is dissolved in 50 µl of IX Sst I solution (50 mM NaCl, 50 Mil, Tris-HCl, pH 7.5, 10 mM MgCl) containing l / 10 units of Sst I enzyme and incubated for 2 h at 37 ° C. The concentration of NaCl is increased to 150 mM by adding 1 µl of 5 M NaCl, 2 µl (U-unit ") of Nde I is added and incubated for another 2 hours at 37 ° C. DNA is precipitated by centrifugation.
Approximately 100 ng of the hydrolyzed plasmid pUCl9 is mixed with 1 pmol of the synthesized binding unit and T4 ligase DNA. Ligase mixture - transform cells E „with 11 K12 JM 109, aliquots are sown on cups i with L-agar containing 100 μg / ml
ampicillin, 40 µg / ml X-gal and
40 µg / ml IPTG
The plates are incubated at 37 ° C. overnight. The colonies containing the plasmid without an insert are blue, and the colonies that contain the plasmid with an insert are white. Several white colonies are selected and selected by restriction analysis for the presence of the Poo 60 fragment. Nde I - Sst I with the same
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Plasmid pCZR336 is a vector carrying the flpL promoter. For the DACS / DAOCS junction with promoter 1 (pL in plasmid pCZR336, synthesized DNA binding must be used. This binding link connects the Nde point of the PCZR336 plasmid with the Sst I point in the DACS / DAOCS coding sequence and has the reduced structure:
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sequence as the synthesized fragment.
The final construction of the plasmid is carried out as follows.
The plasmid is obtained by stitching the Sst I fragment - your HI plasmid with the Nde I - Sst I insert of the p 104 plasmid and the Nde I - Bam HI fragment of the plasmid pCZR336 "Fragment / h / 2.2 t0PooO Sst I - Bam HI plasmid obtained by complete hydrolysis of Bam HI and partial hydrolysis of Sst I. For this, a plasmid of 50 µg is dissolved in 250 µl of IX Sst I solution containing 50 units. Bam HI, and incubated at 37 ° C for 2 hours. Then, 1 µl (10 units) of Sst I 5 enzyme is added and incubated at 37 ° C.
50 μg of the plasmid pCZR336 cyc-i DNA is prepared in 250 μl of a high-salt solution containing 50 units each. enzymes Nde I or and incubated at 37 ° C for 2 h "
Fragment A / 5.8 etc. about. The Nde I-Bam HI plasmid pCZR336, which does not have the LGU coding sequence, is isolated from an agarose gel.
100 μg of the plasmid is suspended in 500 μl of IX Sst I solution containing 100 units. the enzyme Sst I, incubated at 37 ° C for 2 h, the concentration of NaCl is increased to. 150 mM by adding 10 µl of 5 M NaCl, 20 µl (/ 400 food) of Nde I enzyme were added and incubated for 2 hours at 37 ° C. The fragment size / 60 p. O purified in 15% agarose gel.
100 ng of Nde I fragment - Bam HI 5.8 kb Plasmid psgkZb.UO ng fragment 2.2 kb. Sst I - Bam HI plasmid pIT.503 ng fragment 60 p. Sst I - Nde I plasmid0
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Pit 04 is sutured with a T4 pygase and used to transform Eccoli cells. Cells are grown at 30 ° C. Transformants are identified by analyzing their DNA.
Example 3. Determination of E. coli-produced DACS activity / / DAOCS.
Culture E. coli K12 jM 109 / pIT507 for expressing DACS / DAOCS activity
Transformants E. coli K12 JM109 / are grown at 30 ° C overnight in 500 ml of nutrient broth (containing 10 µg / ml of tetracycline) in a rotary incubator, rotating at 250 rpm. The cells are diluted (1:10) and further incubated for 1 hour at 30 ° C under the same growing conditions. The air temperature is then raised to 42 ° C and the incubation is continued for an additional 6.5 hours. Temperature-sensitive from 1857 flpL The promoter, which is set so that it provides for the expression of DACS / DAOCS on a plasmid, is inactivated at 42 ° C and, thus, allows for the expression of DACS / DAOCS. After incubation, cells are harvested by centrifugation and used as the preferred source of E. coli-produced DACS / DAOCS activity.
The activity of expandase and hydroxylase in Eocoli K12 JM109 / cells grown in
Approximately 14 g of Eccoli K12 jM109 / pIT507 cells prepared according to Example 3 were re-suspended in a tris-GEDA solution (15 mM Tris-HCl, pH 7.5, 10% glycerol; 10% ethanol; 10 mM dithiothreitol and 10 mM ascorbate) in a total volume of 20 ml. Cells are sonicated at 4 ° C. During the transmission of sound, phenylmethylsulfonyl fluoride (pMSF) is repeatedly injected until the final concentration reaches 2 mMo. Deoxyribonuclease and magnesium sulfate are added to a concentration of 1 µg / ml and 2 mM, respectively. The suspension is centrifuged at a rate of 40,000 g for 30 minutes. The surface layer contains the raw extract of the enzyme DACS / DAOCS ,,
This extract, 13 ml in volume, was introduced into a 50 ml DEAE-trisacryl column previously equilibrated with 15 mM Tris-GDEA (pH 7.5). Prompt 0
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four volumes of 15 mM Tris-GEDA buffer solution and then a linear gradient from 0.015 mM Tris-GEDA to 0.3 mM Tris-GEDA, 5 ml fractions of 5 ml are collected; the enzyme is eluted as one major peak of activity,
Enzyme activity is determined by performing an analysis based on high pressure liquid chromatography. The expansion-catalyzed reaction is carried out for 15 minutes at 30 ° C using 0.28 mM penicillin N 60 mS-ketoglutarate (L-KG), 0 , 06 mM ferrous sulfate (divalent), 0.67 mM ascorbate, 1.00 mM dithiothreitol, 0.05 mM ATP, and 0.0008 — 0.008 units of enzyme in 1 ml of 50 mM Tris-HCl (pH 7.5. Catalyzed hydroxylase, the reaction is carried out at 36 ° C in the same medium with DAOC with a concentration of 0.05 mM instead of penicillin N.
The reactions are stopped by adding 1 ml of ethanol. The precipitated product is separated by centrifugation at 4000flg for 5 minutes. The surface layer containing the enzyme reaction products is analyzed by high pressure liquid chromatography as follows. Explant activity is determined by measuring the formation of both DAOC and DAC from penicillin H due to the apparent bifunctionality of the enzyme. The hydroxylase activity is determined by measuring the formation of DAC from DAOC.
; Aliquots (20 to 100 µl) of the surface solutions were analyzed for DAOC and DAC using high pressure liquid chromatography (HPLC) using external standards, DAC and DAOC were separated using a column with radially packed compacted microbondpack NH / j. (0.8x10 cm) with a mobile phase of 2% acetic acid, 0.4% methyl alcohol, 6-7% acetonitrile, and 87–92% water (pH 5.8) o The flow rate is 1.5-2.0 ml / min., detection was performed at 260 nm (cephalosporin chromophore). The results of the analysis are reproducible with a 2% deviation in duplicate analyzes catalyzed by both exnase and hydroxylase.
 PRI me R 4 mids.
Plasmid construction (Example 2) contains a genomic, 4 bp, located below the translational endpoint of the DACS / DAOCS coding sequence. In order to reduce the length of this DNA, all but 120 are removed by using the Xa I restriction point located below the translational stop codon at the end of the DACS / DAOCS coding sequence.
10 µg of the plasmid was digested into 50 µl of IX Sst I solution containing LM 0 units of the restrictive enzyme XBa I at 37 ° C for 2 hours.
10 μg of plasmid is suspended in 50 μl of solution IX Sst I with
10, each (from Sst Bam HI enzymes, and incubated for 2 hours at 37 ° C. The end of Bam HI is supplemented with Klenow enzyme.
100 ng of the XBa I fragment (augmented) - Sst I plasmids are mixed with 100 ng of the plasmid digested with Bam HI (augmented) - Sst I, and transformed into K12 jMl 09. The plasmid is identified by restriction enzyme analysis. The plasmid is valuable because the fusion of the complemented ends of XBa I and Bam HI, achieved when constructing the plasmid, reconstructs the Bam HI point (although the point XBa I is destroyed).
50 µg of plasmid is dissolved in 250 µl of high salt solution of 3X containing 50 units. Each of the restrictive enzymes Nde I and Wat III, is incubated for 2 hours at 37 ° C. Fragment l / 191 kb. Nde I - Bam HI is purified on an acrylamide gel 100 ng of a fragment is mixed with 100 ng of a large Nde I fragment - Bam HI of the plasmid pCZR336, ligated and transformed into E. coli K12 JM109. DNA Eocoli K12 JM109 cells can be used as sources of DACS / DAOCS activity according to the procedure described in Example 3.
PRI me R 5. Construction of the plasmid rTG513,
In addition to insertion and deletion mutations, the amino acid composition and sequence of the DACS / DAOC gene product can be changed by replacing the base pair in the protein coding area.
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A known method of point-directed mutation is described in Example 2. The gene of interest is first cloned into, creating a suitable source of single-stranded DNA. 5 μl of M13t18 DNA is incubated in 50 μl of IX Sst I solution containing -10 units. enzyme Bam HI, 2 h at 37 ° С „
50 μg of the plasmid are incubated in 250 μl of IX Sst I buffer solution containing 50 units of each of the Bam HI and Bgl II enzymes for 2 hours at 37 ° C. Tychka Bgl II is located in the 5G-noncoding zone above the initial translational codon of the coding sequence of the DACS / DAOCS vector, and the point. Bam HI is approximately 100 p, o "below the translational stop codon Fragment, 1 ToP.o. Bam HI - Bgl II plasmids are purified as usual in acrylamide.
100 ng of Bant HI hydrolyzed DNA of phage M13 tr18 is introduced into 100 ng of the purified Bam HI - Bgl II plasmid. The mixture is ligated and transformed into an E. col H K12 JM109 Plasmid resulting from the desired insert, has a fragment oriented so that the 5-end DACS / / DAOCS coding sequence (point BglII) is located near the lac Z gene, and the 3 end of this coding sequence (Bam HI point) is located near the lac I gene on the M13 vector. The corresponding isolate is labeled mIT113
The mutagenic seed used for this experiment is synthesized and has the following sequence:, 5 -TCGGACTACTOGCACCTACTATGGCATC-3
Derived from tnIT113, containing the specified sequence instead of the wild-type sequence, identified, characterized in example 2 and denoted as rnIT114. The identification of the desired iso- lite is facilitated by creating a new Alu I restriction point.
10 µg of the mIT114 phage DNA is dissolved in 50 µl of Solution IX, containing 10 units. each of the enzymes Wat I and Nbe I, incubated at 37 ° C for 2 hours. Fragment 1.1 top.o. isolated in acrylamide gel, dislodged from Nbe I-Bam HI with plasmid pCZR336, stitched and transformed into Eocoli,
Transformants E.coll K12 jM109 / pIT51 identified by analysis. Plasmid leads to the expression of a mutant derivative of DACS / DAOCS, which contains a serine group replacing the cysteine group at position 100 in the wild-type E. coli K12 jM109 / p T513 sequence, and is the preferred source of this mutant protein,
PRI me R 6. Construction mid pPS56 and pPS55c
Plasmid pPS56 is an expression vector of Cephalosporium acremonium,. which confers resistance to hydromycin B and contains the Cc.acremoni.urii DACS / DAOCS gene from plasmid pPS56 constructed using the plasmid pMBC12 (NRR B 18097), the plasmid (Example 1) and the plasmid pPS34,
To construct the intermediate plasmid pPS55, 5 µg of the plasmid pMLClZ is dissolved in 5 µl of 1CX Hind III solution and 40 µl of water. 5 µl (50 units of the enzyme. Hind III are incubated at 37 ° C for 2 hours, extracted with phenol and Hind III chloroform, hydrolyzed pMLC12 DNA, precipitated by adjusting the NaCl concentration to 0.25 M and two volumes of cold ethanol, cooled at -70 ° C for 10 min, centrifuged and re-suspended in 5 μl of water
2.3 kb fragment Hind III of the plasmid pPS34 contains the isopenicillin N cephalosporium acremonium promoter associated with the coding sequence of the hydromycin B gene of phosphotransferase, which confers resistance to hydromycin B
10 µg of the plasmid pPS34 is hydrolyzed by the restriction enzyme Hind III, separated on a 0.8% agarose gel, and 2.3 kbp is isolated, the fragment that includes the promoter of the gene Cephalosporium acremonium 1 pS attached and included in the frame of resistance to hydromycin. 1 µg of the fragment was isolated and suspended in 5 µl of water; 1 µl of the hydrolyzed G Hind III DNA plasmid pMLC12 was ligated with 4 µl of the 2.3 kb fraction. Hind III plasmids pPS34 and get plasmid pPS53.
Cells are grown to an optical density of O.D. 5 0 0.5, cooled on ice for 10 minutes, centrifuged. Wasp
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The cells are suspended in 25 ml of cold 100 mM CaCl1, incubated on ice for 25 minutes, centrifuged, the precipitate is re-suspended in 2.5 ml of cold 100 mm and incubated on ice overnight. 200 μl of this cell suspension is mixed with DNA prepared as indicated and incubated on ice for 20 minutes, at 42 ° C for 2 minutes and on ice for 10 minutes. The cells are centrifuged, suspended in 1 ml of nutrient L broth and incubated at 37 ° C for 2 hours.
Aliquots of this cell mixture are plated on L-agar plates containing 25 μg / ml chloramphenicol, 40 μg / ml X- and 40 μg / ml IPTG. The cups are incubated at 37 ° C overnight. Colonies containing plasmid without an insert, such as E. coli K12 JM 109 / pMLC12, show a blue color. Colonies that contain plasmid with an insert, such as E. coli K12 jM 109 / pPS55, show white with clones that contain a fragment of 2.3 kb are selected from several white colonies by restriction analysis of their plasmid DNA. ., Including the Cephalosporium acremonium IPS promoter.
For the final construction of the pPS56 plasmid, 20 µg of the pGHOZ plasmid is hydrolyzed with the Bam HI enzyme and a fragment of 7.0 kb is isolated, including the DACS / DAOCS gene, with 15 µg of the plasmid pPS55 is hydrolyzed with the Bam HI enzyme and the fragment is selected with a size of 5 , 0 kb
The fragments are ligated and used to transform E. coli. Multiple plasmids are identified for the presence of a fragment of the N.O.P., with the binding gene Cephalosporium acremonium DACS / / DAOCS, inserted at a suitable Lam HI point to form the plasmid pPSbb.
Example 7. Construction of plasmid 5 PPSS1.
15 µg of plasmid DNA, C12 is hydrolyzed with an enzyme, and a 2.7 kb fragment is isolated. The resulting fragment is hydrolyzed with the Eco RI enzyme and four DNA fragments are obtained: one, the fragment is the desired molecule L / 2.7 kb, the other is 24 bp long; the third is of length / vO, 6 kb, and the fourth is of length A / 1.9 t „p.
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The acetamidase gene of Aspergillus nidulas was isolated as a restriction fragment 5.0 t, p. EcoRI - Sal I plasmid p3SR2
The reaction mixtures were incubated at 70 ° C for 15 minutes. Both reaction mixtures were incubated at 65 ° C for 10 minutes and then at room temperature for 2 hours and at 4 ° C overnight.
Hind III - Hind III DNA fragment of plasmid pPS51, Bam HI - Hind III - plasmid pIT503 (Example 6); Binding molecules are crosslinked with T4 DNA ligase, E. coli cells are transformed and plasmid pPS52 is obtained by analyzing the presence of a restrictive fragment / 4 7, O t.ooo Bam HI containing the gene Cephalosporiutn acremonium DACS / DAOCS inserted into the Hind III point located between the CAT and amdS genes of the pPS51 plasmid "
PRI me R 9. Construction of plasmids pPS53.
The plasmid pMBS12 (NRRL B-18097) is hydrolyzed with the enzyme Vaga HI. The promoter of the isopenicillin N synthetase gene of Penicillium chrysogenum was isolated as the Nco I-Nco I fragment (1.0 kb) of the plasmid pLC1 °
Single strands of the following binding molecules are synthesized:
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Fragment of size l / 4.3 kb It contains the DNA of the plasmid pBR322, and a fragment with the size of h 5.0 kb. - gene ace tamidase (amdS).
The EcoRI fragment - Sal I of the pMLC12 plamid is ligated with the fragment l / 5.0 t n, o. EcoRI - Sal I plasmid p3SR2 obtained mixture. Transform E.cli.
Identify the colony for the presence of a fragment containing the gene Aspergillus nidulaus and having the exact structure of the desired plasmid.
PRI me R 8. Construction of plasmids RR.
5 μg of the plasmid pPS51 DNA is hydrolyzed with restriction enzyme Hind III,
Single strands of the following binding molecules are synthesized using an automatic DNA synthesizer:
5 -GATCCCCGGG-3
IMI 3 -GGGCCCTCGA-3r
75 pmol of each strand of this binding molecule is individually dissolved in 22.5 µl of water and 2.5 µl of the ligase solution, 1 µl (10 units) of T4 DNA kinase is added, and incubated at 37 ° C for 30 minutes. After reaction







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S -CATGAACAAG-S 1
, If | Ml 3 -TTCTTCCTAG-5
Each single strand of the binding molecule is treated with T4 DNA kinai and then annelated to form an intact binding molecule. 1 μl of the Bam HI hydrolyzed DNA of the pMLC12 plasmid is ligated with 4 μl of the Nco I-Nco I fragment of the plasmid pLCI and 10 μl of the annelated binding molecule.
Ligase mixture transform E. coli. Aliquots of the transformed cell mixture are plated on L-agar plates containing 25 μg / ml chloramphenicol, 40 μg / ml X-gal. and 40 μg / ml IPTG0 Plates are incubated at 37 ° C overnight. Colonies that contain a plasmid without an insert, such as E. coli K12 jM 109 / / pMLC12, show a blue color on
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these cups. Colonies that contain an inserted plasmid, such as E. coli K12 jM 109 / pPS53, show white on these plates. Colonies are selected by analyzing their DNA for the presence of a restriction fragment containing the promoter of the isopene gene.
0 | nicillin N synthetase Penicillium ichrysogenum (plasmid pPS53).
Example 10. Construction of plasmid pPS54.
The gene isaphenicillin N synthetase Re-
5 nicillium chrysogenum was isolated as a Hind III-Hind III fragment (L 3.3 T.Oo) from the plasmid pLC2. 1 μl of the hydrolyzed Hind III DNA of the plasmid pPS51 are ligated with 4 μl of the restriction fragment (3.3 t nn o.) Hind III
plasmid pLC2, producing plasmid pPS54.

Р 11. Construction of plas5
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Plasmid pPS55 was digested with the Bam HI enzyme and a 4.3 kbp fragment was isolated that includes the coding sequence of the HmR gene.
Bam HI - Bam HI - a fragment of the plasmid pPS53 (1 kb in size) containing the gene promoter of isopenicillin N synthetase Penicillium chrysogenum - is ligated with the Bam HI - Wat HI - fragment of the plasmid pPS55, and transformed into E. coli K12 C600 ( ATCC 33524). Identify colonies that contain plasmids with inserts. These plasmids are selected by restriction analysis for the presence of the l / 0.1 kb fragment. Bam HI, which includes the promoter of the PeniciIlium chryso genus IpS gene (plasmid pPS57).
Example 12. Construction of plasmid pPS60.
The plasmid (Example 4) was hydrolyzed with the Bgl II enzyme and ligated with the fragment I, 0 kb. pPS5 plamids (Example 9), transform E. coli K12 0600 (ATCC 33524). Aliquot. This transformed cell mixture is plated on L-agar plates containing 15 μg / ml tetracycline. The cups are incubated at 30 ° C overnight. Colonies that contain a plasmid without an insert, such E. coli K12 0600 / pIT511 are separated from colonies that contain a plasmid with an insert, such as E. coli C600 / pPS60, mainly according to the Eckardt method. Colonies for the presence of a restrictive fragment containing Penicillium chryso- genum IPS gene promoter in the desired orientation (plasmid pRbO).
structures:
5 -CTAGACACCATGACTTCCAAGGTCCCCGTCTTTCGC-3
3 -TGTGGTACTGAAGGTTCCAGGGGCAGAAAGTGGATC-5
Each single strand is individually treated with T4 DNA kinase and then annelated to form an intact binding molecule. The stitched DNA is the desired plasmid pPS59. It is used to transform E. coli K12 C600 (ATCC 33524), Colonies that contain an insert plasmid, such E. coli K12 C600 / / pPS59, are analyzed for the presence of the binding molecule.
Example 15 „Construction of a plamid pPS61,
The plasmid pPS51 DNA (Example 8) is hydrolyzed with the Hind III enzyme and treated with a Klenow fragment in the presence of four dNTPS (dATP, TIP, dGTP and dCTP), precipitated, recovered by centrifugation and re-suspended in 5 μl of water
The plasmid pPS59 DNA is hydrolyzed with the Bam HI enzyme and a fragment of 8.0 tons is isolated,.
The obtained fragment of plasmid pPS59 is treated with the Klenow fragment of DNA polymerase in the presence of four dNTpS (dATP, TTP, dGTP and dCTP) and hydrolyzed with the enzyme Nru I, which leads to the formation of two restrictions
Example 13. Construction of plasmid pPS58
Plasmid pPS60 is hydrolyzed with the enzyme XBa I, which leads to the formation of three DNA fragments; one fragment of 7.9 T. p. and two smaller fragments below 300 p, o. V7.9 kb fragment was isolated. and about 3 μg of the fragment is recovered and suspended in 5 μl of water. 0.5 µg of this fragment is dissolved in 10 µl of a ligase buffer solution, (4 µl) of T4 DNA ligase and 86 µl of water, incubated at 15 ° C overnight. This stitched DNA is a plasmid pPS58, which is transformed with Eccoli K12 C600 (ATCC 33524). TcR colonies are detected, which are analyzed for the absence of two small pieces of XBa I characterizing the plasmid pPS60s
PRI me R 14. Construction of plasmid pPS59c
The plasmid DNA pPS58 is hydrolyzed with the enzyme XBa I and is ligated with the next single strand synthesized
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body fragments: the fragment is 2.1 kb. and a fragment of 5.9 kb. Fragment 2.1 t.PsO. contains the DACS / DAOCS coding sequence under
control of the isopenicillin N gene promoter, Penicillium chryso genum synthetase, and the translational activating sequence. This fragment is ligated. a fragment of the plasmid pPS51
to obtain plasmid pPS61 ,.
Example 16. The construction of plasmids RR
The plasmid pPS57 DNA (Example 11) is hydrolyzed with the Hind III enzyme, which leads to the formation of a single linear 5.3 kb fragment, which is processed with the Klenow fragment of DNA Polymerase 1 in the presence of four dNTpS (dATP, TTP, dGTP and dCTP ). 1 µl of this fragment of the plasmid pPS57 is introduced into 5 µl of the L / 2.1 kb fragment isolated from pPS59 as described in Example 15.
DNA is used for transformation. E. coli K12 C600 (ATCC 33524). Identify colonies that contain plasmids with inserts, with analysis for the presence of a restrictive fragment containing a 2.1 kb insert. (plasmid pPS62).
Example 17, Genetic transformation of Penicillium chrysogeum plasmids p3SR2, pPS52 and pPS54o
The Penicillium chrysogenum strains or any industrial strains obtained from the ATCC strain 9480 by mutation, selection or genetic reproduction for improved production of penicillin G or penicillins V are suitable for use in preparing transformants with vectors and plasmids corresponding to the proposed invention.
The preparation of a homogeneous inoculum for cell culture is carried out as follows.
In order to efficiently transform Penicillium chrysogenum cells, it is necessary to remove cell walls in order to form stable protoplasts. Upon receipt of such protoplasts, it is desirable to begin with a homogeneous inoculum. Otherwise, the production of cells in culture will not be reproducible, and time will be lost when trying to obtain P. chrysogenum protoplasts from inappropriate or insufficient cell numbers.
The ampoule of vegetative cells (P 09 O.K. units in 1.0 ml of preservative medium: lactose 5%, glycerin 10%, and Tween 80 80%) either lyophilized or taken from a storage system in liquid nitrogen and drained at room temperature, diluted with 1.0 ml of sterile saline. 0.1 ml of this suspension is used to inoculate each of the 10 oblique agars among the sporulation, g / l: lactose 15.0; corn extract 2,5; peptone 5.0; NaCl 4.0; MgS047HaO 0.5; KHZPO "}. 0.6; F "Clg; 6HeO 0.005; Si804.-5HO agar 30.0 (pH 7.0) and kept in an autoclave for 20 minutes at a pressure of 120 psi (8.4 kg / cm2) o

Each beveled agar (, 5 cm) contains 25 ml of solidified medium. The inoculum, evenly distributed over the surface of the mown agar, is grown at 25 ° C until it forms and sporulates when mixed with a mesh mycelium (for most strains, 1 week). The product is grown from oblique agar 1 suspended in 10 ml of sterile water.
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- culture mediums, and the suspension is transferred to 106 ml of the aqueous culture medium. The flask containing the suspended cells is placed in a rotary shaking apparatus, and incubated at 25 ° C for 18 hours at 285 rpm with a vertical course of 25.4 mm.
An aqueous culture medium is prepared as follows: 100 ml of solution A, g / l: sucrose 36; L-acnapa- gin 7.5; KNgP04 15; K-jHPO 21; 0.75; MgS04-7Hz. O 0.18; Cac12 0.06; 1 ml / l saline solution (pH of the natural medium) is introduced into a 500 ml shake flask. The flask is closed and maintained in an autoclave at 121 ° C for 20 minutes. Then 2 ml of solution B (glucose 108 g / l) and ml of solution C (sucrose 25 g / l; corn extract - 4% w / v nitrogen) - 12.5 ml; ammonium acetate 5.5 g / l; CaCO 5 g / l; The pH was adjusted to 6.5 with KOH; held in an autoclave at 121 ° C for 20 minutes) was introduced into solution A to obtain an aqueous culture medium.
For the preparation of penicilliun protoplasts, cells from a 24 hour culture were collected by suction filtration (Whatman paper 1 using a Buchner funnel) and suspended in a buffer solution (0.01 M (oxymethyl) aminomethane-5 hydrochloride; 0.01 M MgS04 ; 0.01 M dithiotreitol; 1.00 M KCl and pH 7.0 with HC1).
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Enough buffer solution is injected to a final cell concentration of 1 g of cell mass per 50 ml of buffer solution. This cell suspension is placed in a shaking rotary water bath in a 250 ml shake flask and incubated at 29-30 ° C for 10 minutes at 140 rpm. Dithiothreital-treated cells were centrifuged, re-suspended in 50 ml of enzyme solution (10 mg / ml Novozym, Novoinduscri A / B Bagsvaerd, Dani): 0.1 M tris (oxymethyl) aminomethane chlorohydrate; 0.01 M MgS04, 0.01 M dithio-trentol; 1.00 M KS1 and pH 5.8, in a 250 ml shake flask. This acid suspension is sown in a rotary shaker with a water bath and incubated at 29-30 ° C for 15 min at 140 rpm with vertical
25.4 mm stroke. The treated enzyme cells were centrifuged at 1240 Xg for 6 minutes, the pellet was re-suspended in solution (0.01 M Tris-oxymethyl, 0.01 M MgS04, 1.00 M KC1, pH 7.0). The suspension is centrifuged at 950 Xg for 6 minutes, the precipitate is suspended in the same buffer solution, centrifuged at 700 Xg for 6 minutes, the sediment is suspended again in 5 ml of the same buffer solution. The suspension contains large protoplasts and cells destroyed by osmosis, which retain some cell wall structure. Compared to the small protoplasts removed according to the procedure described, the percentage of protoplasts capable of regenerating cell walls and the percentage of viable osmotically resistant cells are higher for large protoplasts and self-destructible cells in the cell suspension. The cell suspension is diluted with a solution to a concentration of 2x1O8 CLS / ml.
The transformation procedure is as follows.
0.1 ml of the suspension of osmotically destroyed penicillium chrysogenum cells (approximately cl.) Is supplemented with 10 µl of 50 mM CaCl2, 25 µg of plasmid DNA in 5-15 µl of TE buffer solution and 0.9 ml of a solution of freshly dissolved polyethylene glycol 4000. Mixture mix, incubate for 10 minutes at room temperature, centrifuge at 700 Xg for 2 minutes, and mix again. 0.5 ml of the suspension is applied to the surface by an osmotically stable stabilized acetamide medium (1.7 g / l of a yeast nitrogenous base without amino acids and ammonium sulfate; 125 g / l of sucrose, 0.738 g / l of acetamide; 1.27 g / l of CaC and 22 g / l Noble agar). To measure the total number of viable cells present in the transformation mixture, aliquots from this transformation mixture are seeded in plates with a medium in which acetamide is replaced with an equimolar amount of ammonium sulfate. After 7 "- 10 days after the transformation, transfer - mantov colonies are present in the acetamide medium. for reseeding. Abortive transformants are easily separated from resistant transformants, because abortive transformants do not grow when they are replanted into freshly prepared ones.
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acetamide medium. Cells are transformed with a plasmid containing acetates - the das gene from visible colonies, 4-5 days after the transformation,
Analysis of Penicillium chrysogenum / p3SR2 transformant, P ,. chrysogenum / / pPS52 and P, chrysogt: num / pPS54.
The P ° chrysogenim / p3SK2, Pt chrysogenum / pPS52 and P. chry sogenum / / pPS54 transformants exhibit acetaceous - midase activity not found in extracts of the untransformed strain P of chrysogenum (eg ATCC 9480). This activity is a result of the ability of the transformed strains to grow using ammonia released during acetamide hydrolysis, when there is no other source of nitrogen. The resistance of transformants to grow on acetamide as the only source of nitrogen casts the functionality of the acetamidase gene of Aspergillus nidulans in P. chrysogenunu
The transforming phenotype (the ability to grow on acetamide as the sole source of nitrogen) is retained by transformants after passing through a non-selective medium.
Example 18C Genetic transformation of Penicillium chrysogenum with plasmids pPS55 and pPS57t
The preparation of Penicillium chrysogenum protoplasts is carried out in analogy-1, but in Example 1-7,
The transformation procedure was carried out in Example 17c using the plasmids pPS55 and pPS57. Cells transformed with a plasmid containing the hybrid HmR gene produce visible colonies 7–10 days after transformation. The presence of trifluoroperysin in cadmium chloride in the main medium leads to an increase in 5 sensitivity of Ptchrysogenum cells to hydromycin B,
Analysis of P. chryso genum / pPS55 and P. c. Chrysogenum / pPS57 transformants,
Q P, chrysogenum (pPS55) and P chrysogenum / / PS57 exhibit an activity of hydromycin B phosphotransferase that is not found in extracts of non-transformed P. chrysogenum (for example,
- ATCC 9480) “This activity results in the ability of the transfected strains to grow in the presence of toxic concentrations of hydromycin B (with the introduction of hydromycin B in fungal
0
five
49
cells accelerated upon exposure to cadmium ion and trifluoroperazine (TFP). The ability of the transformant to grow in the presence of toxic concentrations of hydromycin B (enhanced by cadmium and TFP) shows that the HmR hybrid gene functions in P „chrysogenum. The higher transformation frequencies obtained with plasmi – Doi pPS57 compared to plasmid pPS55 show that the promoter and the 5 g-regulatory sequences of P. chrysogenum IPS function better in P. chrysogenum than the promoter and 51-regulatory sequences of the Cephalosporium acremonium IPS gene, which is lacking in the pR555 plasmid
Derivatives of the plasmid pPS55 and pPS57 containing the P chrysogenum gene can be used to form penicillin V titers, exceeding the titer of their untransformed-4 parents, thanks to higher levels of IPS production in transfer manta.
Example 19: Genetic transformation of Penicillium chrysogenum with plasmids pPS62 and pPS61.
Preparing the inoculum for glue - an exact culture, preparing the prototype of Penicillium chrysogenum and transformation procedures for using the plasmid pPS62 or pPS61 for transforming P. chrysogenum ana - are logical as described in examples 17 and 18. The plasmid pPS62 contains the HmR gene as selective marker, plasmid PPS61 "- amdS gene as selective mar" Ker.
Analysis of the transformants Pochrysogenum / pPS62 and P.chrysogenum / pPS61.
Persistent P, chrysoge- num / pPS62 and Pochrysogenum pPS61 transformants transforming high molecular weight DNA o This transforming type is retained after passing through a non-selective medium
Plasmids pPS62 and pPS61 contain the DACS / DAOCS hybrid gene, constructed by splicing a template for the Chrysogenum IPS program, with the coding sequence of the Cephalosporium acremonium DACS / DAOCS gene, the natural and hybrid genes encode the synthesis as deacetoxycephalosporin C synthetase, a template, a template, a deacetoxycephalosporin C synthetase, a template, a template, a deacetoxycephalosporin C synthetase, a template, a template, and a deacetoxycephalosporin synthetase gene synthetase, and a synthetase gene, synthesized as deacetoxycephalosporin synthetase, synthetase de synthose, and synthetase synthetase, have developed. porin C synthetase (hydroxylase).
ten
15
20
25
73985650
Enzyme activities are determined accordingly by "- by catalysis of penicillium-N on deacetoxycephalosporin C (expandase) and deacetoxycephalosporic - on C to deacetylcephalosporin C (hydroxylase) p. 61 and P. chrysogenum pPS61 Expansion and hydroxylase activity. These activities were not found in extracts of untransformed P. chrysogenum strains. The promoter and translational activating sequence from the P „chrysogenum IPS gene, the plasmids pPS61 and pPS62 present in the DACS / DAOCS hybrid gene, function in P. chrysogenum, which makes it possible to detect the activity of expandase and hydroxylase
Remark Genetic transformation of Cephalosporium acremonium with the plasmid pPS56o
The Cephalosporium strains or any commercial strains obtained from ATCC 11550 by mutation, selection or genetic multiplication to improve the production of i cephalosporin C are suitable for obtaining neither the transformants by vectors and the mids corresponding to the invention,
The inoculum for cell culture is prepared as follows.
In order to efficiently transform Cephalosporium acremonium cells, it is necessary to remove the cell walls to form resistant protoplasts. When preparing such protoplasts, it is desirable to start with a homogeneous inoculum. In other cases, preparation of cells in culture will not be successful - it will be lost and time will be lost when trying to obtain protoplasts of C "acremonium from inappropriate or sufficient quantities of cells,
To obtain a homogeneous inoculum for cell culture, the ampoule with spores is diluted with 5 ml of sterile saline. About 1 ml of this suspension is used to inoculate 50 canted agar containing 20 ml of trypticase soybean agar (BBL) “Before the inoculation, the medium is dried to dryness. Inoculated-other oblique agars are incubated in
thirty
35
40
45
50
55
for 4 days at 25 ° С, 10-nl of the preservative medium is introduced into the growth medium of the mycelium, which covers the surface of the medium in each canted
e1
agar. The mowed agars are mixed to suspend conidia. Suspensions of conidia from each beveled agar are pooled and 10 ml aliquots frozen at -80 ° C. The frozen conidium suspension slowly loses viability and cannot be used after 3 months of storage at -80 ° C.
Cell growth for the preparation of protoplasts is carried out as follows. 106 ml of aqueous medium in a 500 ml shake flask are inoculated with cells from 10 ml of frozen conidia suspension. Cells are obtained by centrifugation (10 min. 2600 rpm) and suspending in an aqueous culture medium. Before suspension, it is necessary decantation of the surface layer, since lactose and glycerin adversely affect cell growth. The flask containing suspended cells is placed in a rotary shaker with a water bath and incubated at 29-30 C for 24 hours at 285 ob / min. 25.4 mm at 29 - 30 & Co It should be noted that the temperature of 29 ° OOC is different from the temperature (25 ° C), which is preferable for the cultivation of Cephalosporium acremonium for antibiotic dosing.
To obtain protoplasts, cells from a 24-hour culture are collected by filtration with suction (Paper of Watman $ 1 in a Buchner funnel) and suspended in an osmotically stabilized buffer solution (0.8 M NaCl 0.1 M MgS04 and 10 MM. pH 7.0), in which dibothrenol is added to restore the reagent to a concentration of 0.05 M at a final cell concentration of 1 g (after filtration and absorption) of the cell mass per 20 ml of buffer solution. Cycnew is placed with a JB rotary water-bath agitator and incubated at 29 g- 30 ° C for 10 min. 2mer “captoethanol at a final concentration of 40 mM can be used in ka“ -. as a reducing agent. The cells treated with dithiothreitol are collected by centrifugation and re-suspended in solution (t O mg / ml Novorvm 234 from Novo Bio-labs, Bagsvaerd, Dani; 0.8 M NaCl, T), 1 M MgSO 10 IM and pH 5.8) 250 "- a milliliter flask of Erlenmeyer - ka The final concentration is processed

985652
Cell mass is 1 g mass per 10 ml of solution. The cell suspension is placed in a rotary mixer - hivatel with a water bath at 29 "- 30 ° C for 15" - 30 minutes. The suspension of the "- toplasts is subjected to vortex agitation for 2" - 3 s to allow protoplasts to open; zanany with fragments of mycelium. This procedure leads to the formation of a protoplast population
For the transformation procedure for, each plasmid is used. O, 1 ml of suspension containing (1 f 5) X 10 Cephalosporium protoplasts in 0.8 M
ten
15
five
NaCl and 80 M CaClg 20 20 µg of plasmid and polyethylene glycol 4000 were introduced according to Example 17 to a protoplast suspension to obtain a 1.1 ml transformation mixture. This mixture was incubated for 10 min at room temperature and centrifuged0 Then the protoplasts were vortexed in the same fluid. Aliquots (0.1 ml) were applied to the surface of shikazo soy agar medium (BBL), enriched with 10, sucrose to osmotic stabilization of protoplasm
After incubating the Petri dishes at 15 ° C for 24 hours, 4 ml of liquefied agar (0.41% w / v at 42 ° C) containing 0.8 11 NaCl is added to each Petri dish, and Hydromycin B to
5 final concentrations of 100 µg / ml.
Strains C, asgetoplite, exhibiting a low "growth rate as a result of dimensional mutagenesis, are exposed to a reduced amount of
0 romycin during the selection operation (e.g., final concentration 10 µl / ml) o After removing the upper layer, Petri dishes are incubated at 25 ° C in a humidified chamber.
5 Analysis of the transformant Cephalosporium acretnonium pPS56 was carried out in the following way:
The pPS56 plasmid is especially effective for the insertion of multiple
0 copies of the DACS / DAOCS gene in high-molecular weight CephalosptDrium acremoniuni DNA, since it contains the HmR hybrid gene, which functions as the predominant marker in C .asse 5 moniura.
Plasmid pPS56 can be used to create Cepha-1 osporium acremonium strains with cephalospor-1n C, with captions higher than
in untransformed strains, due to copies of the extrachromosomal gene DACS / DAOCSo Copies of the extrachromosomal gene cause higher activities of DAOCS / DACS, which contribute to the psi higher synthesis of cephalosporin, C Strain C asgepuline, producing more quantities of cephalosporin C
and antibiotic production, so that there is only the effect of the above DAOCS activities and: DACS on the synthesis of cephalosporin C.

C. acremonium pPS56 transformants with increased production of cephalosporin C are valuable products for the manufacture of clinically important
 - - W- .-, -, - -s. ± "4l Vx v f AllM. PL - A UftJl Hnfl "
and significant amounts of penicillin. "Cephalosporin antibiotics, such N, are valuable parental ones, like cephalotin sodium, cefamandolnaphate strains for transformation using cephazolin sodium,

权利要求:
Claims (1)
[1]
by claiming a plasmid pPS56 for the purpose of the Formula for the Invention of Transformant Transformations, which are more than a Method of constructing a recombine effectively transform Penicillium N t, Nantes Plasmid DNA encoding to cephalosporin C. Desired transfer - deacetoxycephalosporin C enzyme
synthetase / deatsetshchefalosporin C synthetase with the following amino acid sequence:
Ccacrtsnonium / pPS56 manty carry transforming DNA as inserts in neutral zones in terms of root
H2N-MET.THR SER LYS VAL PHO VAL PHE ARC LEU
TYIR GLU LE GLA GLU LE GLA GLA BLU GLA SER GLA SER GLU GLU GLU GLU LYS ARC ALA VALA THR LEU ALA ASPA ARC ASD ALA ARC ARC GLY PHA SER ALA LEU GLU TRA GLU IRA GLA IRA PHE PRO ASN ARC GLY PHE GLU ASP VAL TRP GLN ASP TYR. PHA ASP ARC META GLY ALA ALA PROPERTY GLA-GLUA ASP ILE ASPA ARC VAL ALA GLU GLU GLU PRO
i
LEU SER THU ILE THR LEU VALIUS I GLA THY ALA CYS ALA ISTVA GLY GLY LYS VL LYS LALA PRO LYS HY ROSE ARG VALA LY LY SER PRO fel arg gl gl se arg gl gl se g r u r l a r l a r l a r l a r l a r l a r t l r w COOH.
that is, the lead or the Penicillium fragment with the fragment leads to the DNA fragment encoding 55DNA encoding DAOCS / DACS with the following sequence of the upstream nucleotide sequence of the promoter or gene by isopenicillinity: N-synthase Cephalosporium acremo and antibiotic products the place is only the influence of the above activities of DAOCS and: DACS on the synthesis of cephalosporin C.
C. acremonium pPS56 transformants with increased production of cephalosporin C are valuable products for the manufacture of clinically important
 PL - A UftJl Hnfl "
cephalosporin antibiotics such as cephalotin sodium, cefemandolnaphate and cefazolin sodium,
&
 ACT TSS ALS-OTO CCC G-TC TTT CGT STO,
OJSC SLS STO LAS LSS GGC LAS-OTO STO ACC GAG STO GCC GAG-GCC GTC ACC ACC AAG-GGT ATC TTC TAG TTO-LSS GAG-AGC GЈC STO G-TC GAC JSC1
GAC; CAC ACC tcc-GCC-CGT GAG ACG TGC GTT GAC ttt tts AAG ms GGA
ACC GAG-GAG-GAG AAG-ACG GCC GTG ACG-CTC GCCGAC CGT AAC GCC COG
CGC GGC TTC TOT GCC CTC. GAG TGG GAG AGC LCC GCC GTC GTC ACC GA &
ACG- GGC AAG TAG TCG GAC TAG TOO AGO TGC TAGTOO ATG GGC ATC GGC
GGC AAC CTG .TGS CCG AAC CGG CCC TTC GAG GACG2C TGG GAG GAC TAG
TTC GAC CGC ATG TAG GGC GCA GCC AAG GAT GTC GGC CGC GCC GTT CTC
AAC TCT GTG GGC GCC CCG CTC.GCC G € G GAG GACATT GAT GAC TTC GTC
GAG-TGC CAT GCC CTC CTC CGC CTA CGG TAG TTCCCC GAA GTG CCC GAG-GAG CGC GTC GCC GAA GAG GAA CCC CTC CGC ATG GGA CCC SLS TAG GAC
t
CTA. TCG-LSS ATS ACG CTC GTG GAG GAG AGA.GCC TOG 6CC AAC GOG TTC GTG AGC CTG-GAG TGG GAG GTC TGG GGA GAA TTC GTC GTC GTC GTC GGC LSS STO 0CC ACG-GGC GGC AAG-GTC AL6-GCG CCC AAG-GAG CGG GTC AAG TCT CCC GGO CGC GAC CL0-CCG GTC GGC AGC CGC ACG TCG-AGC GTC TTC .TTC CTG-CCC. CCG ALS CCG GAC TTC AGC TTC ALS GTG CAG-. GAG TCG AGG- OJSC TGG-GCT TTC AAC GTC CGC ATC CCG TCG GAG CGC AGO. ACG-TTC AGG-OJSC T09-CTT GGC GGG-AAC TAT G-TC AAC ATG CGG-AGG GAT AAG-CCG GCG GCA GCG-CAC. GCG CCT GTC CCC GCG GCT GCC CGT GTC TCT ACC GCA ACCA GCT GCT
G5GЈGAST3 with a DNA fragment coding for 3 -ter-Penicillium chrysogenum or Cepha-terminal sequences of the gene losporium acremonium followed by
isopenicillin N-synthetase Cephalos-45 selection of clones containing plasmids
porium acremoniutn or Penicillium. pTT507 or PLT511, or PPS58, or
and a fragment from the genetic mark-pPS359, or pPS60, or pPSol, or
rum TSG, or amds, or Hmr, transformer pPS62, or RC, 502, or pPS52, or
the resulting recombinant pPS56.
DNA of Escherichia coli strains or
Compiled by T. Zaboykina Editor I. DerbakTehred M. Didyk Proofreader L. Patay
/.
Order 2013 Circulation Subscription
RCIIPI State Committee for Inventions and Discoveries at the State Committee on Science and Technology of the USSR
113035, Moscow, Zh-35, Raushsk nab. 4/5
..- ™ ™ ™ -in MI -. - - (((in - - GG-PGv-in "in 11 P11 PG IYaITI Production and Publishing Combine Patent, Uzhgorod, Gagarin str., 101

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法律状态:
2007-12-20| REG| Reference to a code of a succession state|Ref country code: RU Ref legal event code: MM4A Effective date: 20060304 |

优先权:

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申请号 | 申请日 | 专利标题

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US2183687A| true| 1987-03-04|1987-03-04|

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