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    • 专利摘要:
    Recombinant protein from Nannochloropsis gaditana. The present invention relates to the use of a recombinant protein as an inhibitor of tumor growth, specifically to the use of the recombinant protein UCA01, which is a protein that immerses the cell in a period of mitotic inactivity in the case of tumor cells. In this way, it prevents tumor growth, in addition to preventing its appearance. UCA01 prevents tumor development due to three factors, its antioxidant capacity, increased activity of the p53 protein, and protection from mitochondrial degradation. Specifically, in the present invention the biological activity of the recombinant UCA01 protein extracted from the microalga (N. gaditana) is demonstrated for the first time. It is noted that the wild-type UCA01 protein was, prior to the present invention, initially classified as "hypothetical protein" in the NCBI (Accession: XM_005854224.1). (Machine-translation by Google Translate, not legally binding)
    公开号:ES2810229A1
    申请号:ES201930775
    申请日:2019-09-05
    公开日:2021-03-08
    发明作者:Acero Francisco Javier Fernandez；Vallejo Roberto Andres；Reinado Rafael Carrasco；Garcia Palmira Guarnizo；Quinones Carlos Fajardo；Cobos Lidia Tomas；Romani Laura Soriano；Lopez Victoria Capilla；Nino Almudena Escobar
    申请人:AINIA；Neoalgae Micro Seaweeds Products S L；Universidad de Cadiz；Endesa Generacion SA；
    IPC主号:
    专利说明:

    [0002] RECOMBINANT PROTEIN FROM GADITANA NANNOCHLOROPSIS
    [0003] TECHNICAL SECTOR
    [0004] The invention belongs to the field of biomedicine, in particular to the use of a recombinant protein of N. gaditana also known as Microchloropsis gaditana (Fawley, Jameson, & Fawley, 2015), which, when purified, has an anti-proliferative effect on tumor cells .
    [0006] BACKGROUND OF THE INVENTION
    [0007] Cancer therapy has undergone major breakthroughs that have occurred with unprecedented speed. The therapeutic arsenal has been intensified in efficacy by counting on cytostatics or what is commonly known as "chemotherapy", drugs with different mechanisms of action that induce apoptosis or cell death but have little specificity to exclusively attack the tumor cell, also affecting healthy cells. New therapies directed against a specific target, a protein or a membrane receptor located in the tumor cell have recently appeared. The first anti-target therapies introduced were monoclonal antibodies administered intravenously, however, in recent years we have witnessed a true "boom" with the development of a new family of oral targeted therapies, called tyrosine kinase inhibitors. These novelties in support of surgery and oncorradiology with their spectacular advances in recent years have contributed significantly to an improvement in the control of the disease or in the quality of life of patients.
    [0009] Even so, there is still a need to provide new therapies that induce apoptosis or cell death or prevent tumor growth, and that have high specificity against tumor cells.
    [0011] BRIEF DESCRIPTION OF THE INVENTION
    [0013] The present invention relates to the use of a recombinant protein as an inhibitor of tumor growth, specifically to the use of the recombinant protein UCA01, which is a protein that immerses the cell in a period of mitotic inactivity in the case of tumor cells. In this way, it prevents tumor growth, in addition to preventing its appearance. UCA01 prevents tumor appearance due to three factors, its antioxidant capacity, mitochondria. Specifically, in the present invention the biological activity of the recombinant protein UCA01 extracted from the microalga ( N. gaditana) is demonstrated for the first time. It is noted that the wild-type UCA01 protein was, prior to the present invention, initially classified as "hypothetical protein" in the NCBI (Accession: XM_005854224.1).
    [0015] Specifically, the authors of the present invention have shown, for the first time, that the recombinant UCA01 protein obtained from N. gaditana reduces the cell growth of tumor cell lines (HepG2 and Caco-2) without affecting the growth of the non-tumor cells tested (Ea.hy926). This inhibition is also directly proportional to the concentration of UCA01 tested in the case of using the colon adenocarcinoma cell line, so that, as the concentration of the protein increased, the greater the inhibition of tumor growth. However, this effect was not observed on the growth of the non-tumor cell line used (Ea.hy926). Therefore, it is deduced that the recombinant N. gaditana protein UCA01 of the invention has a selective anti-proliferative (cytotoxic) effect since it only acts on the tumor cells tested.
    [0017] Finally, it is important to emphasize, by virtue of the aforementioned, that the activity of the recombinant protein of the invention (UCA01) as an antitumor provides a tool to fight cancer, based on a protein from a microalgae. This inhibition takes place at low concentrations, compared to other biological activity assays in other proteins, which indicates a new method and a new source for obtaining the efficient UCA01, since the amounts necessary to carry out its antitumor activity have been minimal.
    [0019] BRIEF DESCRIPTION OF THE DRAWINGS
    [0021] Figure 1: Functional domains of prohibitin 1 (Theiss & Sitaraman, 2011).
    [0023] Figure 2: (a) Primers designed for the amplification of the complementary DNA of N. gaditana , with the restriction enzymes used to create sticky ends with the plasmid pET28a; (b) Confirmation of Agarose gel amplification; (c) Confirmation of sequencing by 100% Blast homology with UCA 01 homologs; (d) Matrix of percentage of identity, created by Clustal 2.1, firstly, Protein UCA01, compared sequencing of NCBI: with prohibitin from N. gaditana and several of human prohibitins.
    [0024] E. coli bacteria, Rosseta gami (DE3).
    [0026] Figure 4: Image of the 10% SDS-PAGE gel from the UCA01 purification process.
    [0028] Figure 5: Antiproliferative effect of the recombinant protein UCA01 from N. gaditana. The effect on cell lines was evaluated: HepG2 (liver carcinoma) / 24 hours; Caco-2 (colon adenocarcinoma) / 24 hours and EA.hy926 (endothelium) / 6 and 72 hours. The mean ± standard deviation of the mean (SEM) is represented. Statistic used: tstudent comparing each value with the control with DMSO.
    [0030] DESCRIPTION OF THE INVENTION
    [0032] Definitions
    [0033] A "recombinant protein" is a protein that has been produced in a host cell, of a species other than the original cell, that has been transformed with the nucleic acid encoding the protein.
    [0035] The term "inhibit proliferation" refers to any method that decreases the capacity for cell growth.
    [0037] "Prohibitin", also known as PHB, is a protein that in humans is encoded by the PHB gene. The PHB gene has also been described in animals, fungi, plants, and single-celled eukaryotes. Prohibitins are divided into two classes, called Type I and Type II prohibitins, based on their similarity to the yeast genes PHB1 and PHB2, respectively. Every organism has at least one copy of each type of prohibitin gene (Mishra, Murphy, & Murphy, 2006; Van Aken et al., 2007).
    [0039] The term “microalgae” refers to microscopic algae, generally found in fresh and marine water systems, living both in the water column and in sediments (V.thurman, 2007). They are unicellular species, which can be organized in chains or groups. Depending on the species, their sizes can vary from a few micrometers (^ m) to a few hundred micrometers (Thrush, Hewitt, Gibbs, Lundquist, & Norkko, 2006). Microalgae, capable of photosynthesis, are important for life on earth; they produce about half of the atmospheric oxygen and simultaneously use the carbon dioxide from the greenhouse gas to grow photo they provide energy for all trophic levels above them (Thrush et al., 2006).
    [0041] DESCRIPTION
    [0043] RECOMBINANT PROTEIN OF THE INVENTION
    [0045] The authors of the present invention have shown, for the first time, based on the data shown by the NCBI, that the recombinant protein UCA01 extracted from microalgae ( N. gaditana), classified up to now as "hypothetical protein" (hypothetical protein) in the NCBI (Accession: XM_005854224.1), it shares common domains with the proteins of the SPFH superfamily (stomatin, prohibitin, flotillin, and HflK / C) (Figure 2), specifically with prohibitin. However, said homology of the UCA01 protein is reduced to only 56.36% (clustal W) with the human homologous prohibitin (figure 2), which, a priori, would not allow to deduce that it had the same functions as human prohibitin . In fact, when it was compared with several human prohibitins (GenBank: S85655.1, GenBank: L14272.1, GenBank: BC095460.1, GenBank: BT007411.1) the homology found was respectively 56.34% and 56.22 %, still small percentages that, again, did not allow to deduce that it had the same functions as human prohibitin. Therefore, in order to determine and clarify these functions, the recombinant protein UCA01 was extracted from the Nannochlompsis gaditana Lubian CMP 572 microalgae (Fernández-Acero et al., 2018), cloned and purified. Specifically, the recombinant UCA01 protein of the present invention, which has the nucleotide sequence SEQ ID NO 1:
    [0047] SEQ ID NO 1:
    [0048] ATGTCTCCAGCAGGACCGCTGGGAGCCTTGCTAGGTGTGACGGGCATCCTTTACGCCGG GTACAATAGTTTTTACACGGTGGAGGGTGGGCACCGAGCTCTGTTGTTCAACAGATTAATC GGTGTGAAAGAAGAAGTGTACATGGAGGGAATGCATTTTATGATTCCCTGGTTCGACATGC CCATCATTTACGACATCCGCCCCAAGCCCCGGATGATCCAGTCCTTGACAGGAAGCAAAG ACATGCAAATGGTCAACATCACCATCCGCGTTTTGTCTAAGCCCGACTCGGCTCAACTCCG CTGGATCTTCCGCACCTTGGGTCGCGACTACGACGAGCGTGTCCTCCCTTCCATCGTCAA CGAGGTCTCCAAGGCCGTGGTGGCCAAGTACAACGCCGCGGAGCTCTTGACGAAGCGTG AGATGGTCTCCACCCAAATCCGGTTGCAGTTGGAGAAGCGTGCGAAGGAGTTTCGGATCG TCCTGGACGACGTGTCGATCACCCACTTGACCTTCTCCCGGGAGTACACGAACGCGGTCG AGGCCAAGCAAGTTGCTCAACAGGAAGCCGAGCGCGCGAAATATGTGGTAATGAAAGCGA ACCAAGAAAAGGAAGCCATCATTATCAAAGCGGAGGGAGAGGCCCAATCCGCTGCTCTGG TGGGTAAGGCCATTCGAGAGAATCCTGCTTTTATCAAGCTGCGCAAAATCGACGCAGCCA TCTTGTTGAATATGTACTCGGGCGACGAAGAGAAGTCTGGAAAGAAGCGGTAGGCGGCCG CACTCGAGCACCACCACCACCACCAC,
    [0050] from which the following amino acid sequence was obtained (SEQ ID NO 2):
    [0052] MSPAGPLGALLGVTGILYAGYNSFYTVEGGHRALLFNRLIGVKEEVYMEGMHFMIPWFDMPIIY DIRPKPRMIQSLTGSKDMQMVNITIRVLSKPDSAQLRWIFRTLGRDYDERVLPSIVNEVSKAVVA KYNAAELLTKREMVSTQIRLQLEKRAKEFRIVLDDVSITHLTFSREYTNAVEAKQVAQQEAERAK YWMKANQEKEAIMKAEGEAQSAALVGKAIRENPAFIKLRKIDAARDIANWSSSGQKVYLSADS LLLNMYSGDEEKSGKKRAAALEHHHHHH,
    [0054] It was synthesized from the Nannochlompsis gaditana Lubian CMP 572 microalgae (Fernández-Acero et al., 2018). For this, once the proteome of this microalga was analyzed, primers were designed to amplify the desired area of the complementary DNA region, thus avoiding amplifying the exons present in the original DNA sequence of N. gaditana. Subsequently, the complementary DNA was ligated with the Pet-28a plasmid using the cutting enzymes Ndel and SalI, thus joining the plasmid together with the complementary DNA, being introduced into the Top10 strain of E. coli, used as a storage strain of construction.
    [0056] Once it was confirmed by PCR in the E. coli strain Top10, the fragments obtained were isolated and sequenced. The sequence thus obtained presented 100% homology and coverage with the "hypothetical protein" in the NCBI (Accession: XM_005854224.1) of N gaditana (figure 2). This construction, present in the Top10 strain, was used to transform the strain Rosetta gami from E.coli, to be used as an expression strain, (DE3) UCA01 protein was expressed using IPTG as an expression induction method and confirmed on an acrylamide SDS-page gel (Figure 3).
    [0058] Following production of the recombinant protein in the host cell, the cell lysate was passed through an affinity column for purification. The fusion protein with the tag was retained on the column while the other proteins and other contaminants were flushed through. That is, this protein was purified with His Spin Trap affinity columns (GE Healtcare), thanks to the N-terminal polyhistidine tail (6 histidines) present in the Pet-28a plasmid and added to the protein.
    [0060] From said cloning and purification, the authors of the present invention demonstrated, for the first time, the antitumor activity of this recombinant protein of N. gaditana in the line it inhibited the proliferation as well as the cell viability of these tumor lines. Furthermore, this effect maintains a correlation directly proportional to the concentration of recombinant UCA01, where the higher its concentration, the greater the inhibition, or what is the same, the lower the cell viability of the tumor line.
    [0062] Therefore, a first aspect of the invention refers to a recombinant protein obtainable by a process comprising:
    [0064] a) integrating an insert with a nucleotide sequence comprising or consisting of SEQ ID NO. 1, in a genetic construct or an expression vector,
    [0065] b) optionally, transforming a host with the genetic construct or with the expression vector of step (a), where said host is a storage strain of the construct,
    [0066] c) transforming a host with the genetic construct or with the expression vector from step (a) or step (b), where said host is an expression induction strain, d) inducing the expression of the recombinant protein,
    [0067] e) extracting the recombinant protein, and
    [0068] f) optionally, purify the recombinant protein
    [0070] It is noted that step a) set forth above can alternatively be carried out by inserting a variant nucleotide sequence exhibiting an identity of at least 80% with SEQ ID NO: 1.
    [0072] The term "identity" as used herein refers to the ratio of identical amino acids or nucleotides between two amino acid or nucleotide sequences being compared.
    [0074] The term "homology", as used herein, refers to the similarity between two structures due to a common evolutionary ancestry, and more specifically, to the similarity between the amino acids of two or more proteins or amino acid sequences.
    [0076] Since two proteins are considered homologous if they have the same evolutionary origin or if they have similar function and structure, it is generally assumed that values greater than 30% similarity or identity indicate homologous structures. We can therefore consider that identity percentages of at least 80% will include the conserved active regions.
    [0077] step a) will present an identity of at least 90% with SEQ ID NO: 1. In another more preferred embodiment of this aspect of the invention, the nucleotide sequence of step a) will present an identity of at least one 95% with SEQ ID NO: 1. In an even more preferred embodiment of this aspect of the invention, the nucleotide sequence of step a) will have an identity of at least 98% or 99% with SEQ ID NO : one.
    [0079] The design of the expression vector or the gene construct of the invention is based on genetic engineering techniques, where said expression vector or the gene construct of the invention may comprise, in addition to the nucleotide sequence SEQ ID NO. 1 or any variant thereof, elements that regulate the expression of said sequence. Such regulatory elements include promoters and enhancers. Promoters are typically positioned 5 'to the transcription or translation initiation site. Enhancers are capable of influencing gene expression when they are in position 5 'or 3' with respect to the cDNA or when they are part of an intron. Regulatory sequences include, in addition to promoters, sequences that facilitate translation, processing signals for introns, stop codons, signal sequences, internal ribosome binding sites (IRES), and polyadenylation signals.
    [0081] The expression vector or gene construct of the invention comprising the nucleotide sequence SEQ ID NO. 1, must be operatively coupled with a sequence that regulates the expression of said nucleotide sequence SEQ ID NO. 1. The person skilled in the art will realize that the type of vector suitable for the expression of the nucleic acids and gene constructs of the invention will depend on the organism in which it is desired to express the polynucleotide of the invention.
    [0083] In a preferred embodiment of this aspect of the invention, the expression vector is the prelinearized vector pET28-Luci.
    [0085] A second aspect of the invention relates to host cells or organisms as expression inducing strains comprising the expression vector or the gene construct, as defined in the invention. In principle, any type of host organism known to the person skilled in the art can be used in the present invention, such as a bacterial strain ( Escherichia coli, Bacillus subtilis and the like), a yeast strain ( Saccharomyces cerevisiae, Pichia pastoris, Kluyvemmyces lactis, Hansenula polymorpha and the like), a transgenic plant (dicotyledonous or monocotyldonean), an insect cell, for example baculovirus, a mammalian cell (COS, CHO, C127, HeLa cells rabbit, a pig, etc.).
    [0087] In another preferred embodiment of this aspect of the invention, the expression bacteria are chemically competent Eschenchia coli cells, possessing a suitable genotype for the transformation and expression of proteins, and whose genome is known (Genome sequences of Escherichia coli B strains REL606 and BL21 (DE3). Jeong H, et al. J Mol Biol 2009 Dec 11).
    [0089] A competent bacterium is characterized by having a weakened bacterial wall and therefore has an easier time capturing foreign DNA through a process of thermal or electrical shock (transformation). Expression bacteria are used for the production of the protein. In this specification, the expression bacteria are those that possess the machinery necessary to overexpress the inserted cDNA and produce the recombinant protein.
    [0091] In another preferred embodiment of the first aspect of the invention, the integration of the nucleotide sequence SEQ ID NO: 1 of step (a) is carried out by means of a ligation process.
    [0093] To carry out the ligation process, the insert: plasmid mixture was resuspended in the In-Fusion Dry-Down pellet product (Clontech). In-Fusion Dry-Down pellet is a lyophilisate that contains the In-Fusion enzyme, which favors the attachment of the insert to the plasmid thanks to the homology in the nucleotide sequence present in both.
    [0095] Therefore, in another preferred embodiment of the invention, a lyophilisate comprising the enzyme In-Fusion is used in the ligation. This is a Poxvirus DNA polymerase with 3'-5 'exonuclease activity, which is capable of binding single-stranded DNA molecules that have short, homologous sequences at their ends, such as an amplified PCR product and a vector.
    [0097] In another preferred embodiment, the insert of step a) of the first aspect of the invention is synthesized using the nucleotide sequence primers SEQ ID NO: 3 and SEQ ID NO: 4.
    [0099] A third aspect of the invention refers to a protein comprising the amino acid sequence SEQ ID NO: 2, or to the recombinant protein of the invention, for use as a medicine; or, alternatively, to a protein comprising the amino acid sequence SEQ ID NO: 2 or to the recombinant protein of the invention for use in the manufacture of a medicine.
    [0100] amino acid sequence comprising or consisting of SEQ ID NO: 2, but also variants thereof, which have an identity of at least 90% with SEQ ID NO: 2. Preferably, they have an identity of at least 95% with SEQ ID NO: 2. More preferably, they have an identity of at least 98% or 99% with SEQ ID NO: 2 (hereinafter these variants will be referred to as "variants of the recombinant protein of the invention").
    [0102] A fourth aspect of the invention refers to a protein comprising the amino acid sequence SEQ ID NO: 2 or variants thereof, for use in the treatment of cancer or the treatment of disorders of unwanted cell proliferation, or a antiangiogenic drug.
    [0104] A fifth aspect of the invention is constituted by a method for the treatment of cancer, cell proliferation disorders and unwanted angiogenesis, in which a pharmaceutical composition comprising an effective amount of at least one protein comprising or consists of the amino acid sequence SEQ ID NO: 2 or variants thereof, to an individual in need. The administration of said pharmaceutical composition constitutes a method for treating or preventing a solid tumor in a subject, such as a tumor or colorectal cancer, esophagus, lung, prostate, breast, pancreas and liver, where the method comprises the administration of a composition comprising a protein comprising or consisting of the amino acid sequence SEQ ID NO: 2 or variants thereof in an amount effective to slow or block tumor cell growth. In one embodiment of the invention, the composition can be administered to patients with solid tumors, such as a tumor or colorectal cancer, esophagus, lung, prostate, breast, pancreas and liver, blocking the proliferation of the cancer cell, even in presence of inflammatory stimuli.
    [0106] Additionally, the data provided in this invention indicate that the recombinant protein of SEQ ID NO 2 is effective in blocking cancer cell growth. More specifically, the results demonstrate that the recombinant protein facilitates cancer cell death. That is why the invention also provides a method for the inhibition of the development of tumors associated with inflammation and their metastases, which comprises the administration of the recombinant protein to a subject in need. Among the tumors associated with inflammation and their metastases are, for example, the following types of cancer: colorectal, esophagus, lung, prostate, breast, pancreas and liver.
    [0107] prophylactically, to prevent the development of cancer associated with chronic inflammation, such as Crohn's disease, ulcerative colitis, pancreatitis, cirrhosis, etc. Therefore, it is also an object of the present invention, a method for the prevention of cancer associated with chronic inflammation characterized in that the recombinant protein of SEQ ID NO 2 or a variant thereof or a composition comprising said protein is administered to an individual in need.
    [0109] Regarding the dose and treatment scheme to be followed with the compositions comprising the recombinant protein of SEQ ID NO 2 or variants thereof, a person skilled in the art can easily determine the dose and treatment scheme (prophylactic or therapeutic). Effective amounts can vary depending on the relative potency of individual compositions, and can be calculated based on the molecular weight of the recombinant protein in vitro or in animal studies. For example, given the compound's molecular weight (chemical structure) and an effective experimental dose (IC50), a dose in mg / kg of weight can be routinely calculated. In general, the doses are 0.2-4 mg / Kg of weight.
    [0111] On the other hand, a sixth aspect of the invention relates to pharmaceutical compositions comprising at least one protein comprising the amino acid sequence SEQ ID NO: 2 or variants thereof, and pharmaceutically acceptable excipients or carriers.
    [0113] The invention also provides compounds for cancer diagnosis comprising at least one of the proteins comprising the amino acid sequence SEQ ID NO: 2 or variants thereof, and an imaging agent selected from the group consisting of a fluorescent group, a non-fluorescent group, a fluorescent semiconductor particle, a paramagnetic or superparamagnetic agent, and a radioisotope.
    [0115] Another aspect of the present invention is a pharmaceutical combination comprising at least one of the proteins comprising the amino acid sequence SEQ ID NO: 2 or variants thereof, with at least one treatment agent, such as anticancer drugs and hormones. In one embodiment of the invention, in said combination said proteins are directly conjugated to the treating agent through covalent bonds. In other cases, the protein is conjugated to the treating agent by a coupling element.
    [0117] The invention also encompasses pharmaceutical combinations where at least one of the proteins comprising the amino acid sequence SEQ ID NO: 2 or variants thereof, is combined with any type of chemotherapeutic or radiotherapy treatment. The elements in the course of medical treatment, sequentially or simultaneously. In one embodiment of the invention, said specific drug for standard chemotherapy is selected from cisplatin and 5-FU. In said pharmaceutical combination, the agents and drugs that are part of it can be administered simultaneously, separately or sequentially in the course of the same treatment.
    [0119] Any of the proteins, compositions of any of the aforementioned aspects can be administered by any route of administration including parenteral, for example: intravenous, intramuscular, or subcutaneous injection, as well as topically to achieve their therapeutic effectiveness.
    [0121] The following examples serve to illustrate the present invention but should not limit it.
    [0123] EXAMPLES
    [0125] Experimental development.
    [0127] Once the amplification of this protein had been verified, the recombinant protein was made, following the following steps.
    [0129] Isolation of total RNA from N. gaditana
    [0131] Total RNA was isolated by NucleoSpin RNA® (Macherey-Nagel) following the manufacturer's instructions. In summary. The quality of the isolated RNA was verified, following the indications of the Agilent RNA 6000 Nano © Kit.
    [0133] Synthesis of complementary DNA by RT-PCR
    [0135] To carry out this procedure, the indications of the commercial qScript® kit (Quanta Biosciences) were followed. The reaction protocol used was the following: 1 ^ L of the isolated RNA (1-10 ^ g of total RNA), 14 ^ L of nuclease-free water, 4 ^ L of the qScript reaction mixture (5X) and 1 ^ L for qScript reverse transcriptase; for a final volume of 20 ^ L. It was gently vortexed and then centrifuged for 10 sec. Subsequently, they were placed in a thermal cycler under the following program, 1 cycle at 22 ° C for 5 min., 1 cycle at 42 ° C for 30 min., One cycle at 85 ° C for 5 min. with a stop at 4 ° C.
    [0136] PCR amplification
    [0138] For this procedure, the indications of the commercial kit Phusion Flash High-Fidelity PCR Master Mix (Thermo Scientific) were followed. The reaction conditions were as follows: 25 j L of 2X Phusion Flash PCR Master Mix, 2.5 j L of the primer 10 jM Prohi-F: 5'-GGCATATGTCTCCAGCAGGACCGCTGG-3 '(cleavage site for Ndel) (SEQ ID NO 3), 2.5 | jL of the first 10 jM Prohi-R: 5'-GGGTCGACCTACCGCTTCTTTCCAGACTTC-3 '(SalI cleavage site) (SEQ ID NO 4), 2 j L cDNA (65 ng / jL) and 18 j L of water, for a total reaction volume of 50 L. with j amplification with primers Prohi-F and Prohi-R 834 bp fragment (GenBank XM_005854224.1) is obtained. The thermal cycler program was as follows: initial denaturation at 98 ° C for 10 sec, followed by 30 cycles at 98 ° C for 1 sec, 72 ° C for 5 sec, 72 ° C for 15 sec, a final extension cycle at 72 ° C for 3 min and stop at 4 ° C. The amplified product was visualized by means of a 1% agarose gel run for 1 h at 110 V and stained with Gel-Red (figure 2).
    [0140] Purification of the amplified fragment.
    [0142] This procedure was carried out following the instructions in the commercial GeneJET Gel Extraction Kit (Thermo Scientific). For this, the following protocol was followed: once the band had been cut, it was weighed and a volume of Binding Buffer was added in relation to the weight of the band (1: 1 ratio, weight: volume). Subsequently, the mixture was incubated at 60oC until the gel band was completely dissolved. The solubilized gel solution was then transferred to a GeneJET purification column, centrifuged for 1 min (12,000 rpm), and the resulting liquid was discarded. Then 700 µL of the Wash Buffer was added to the column, it was centrifuged for 1 min (12,000 rpm) and the resulting liquid was discarded. It was centrifuged again for 1 min (12,000 rpm) to eliminate the residues of the Wash Buffer. Elution was performed with 40 j L Elution Buffer, allowed to incubate for at least 1 min and the centrifuged for 1 min (12,000 rpm). The fragment purified from the gel bands was visualized by means of a 1% agarose gel run for 1 h at 110 V and stained with Gel-Red.
    [0144] Restriction enzyme digestion.
    [0146] In order to linearize the vector pET28a (Novagen) to link it to the UCA01 fragment, the following reaction was carried out: 7 ^ l of pET28 (2 ^ g), 10 ^ l of 10X Buffer D, 2 ^ l of Ndeí (10 U / ^ l), 2 ^ l of Salí (10U / ^ l) and 79 ^ l of water, for a total reaction volume of 100 ^ l. It was incubated for 4 h at 73 ° C. Then 11 l of 10X FastAp buffer, 2 l of alkaline phosphatase (1 U / ^ l) were added and incubated for 1 h at 37 ° C. With the UCA01 fragment, the 10X Buffer D, 2 ^ L of Ndel (10U / ^ L), 2 ^ L of SalI (10U / ^ L) and 66 ^ L of water. Total reaction volume of 100 ^ L. It was incubated for 4 h at 37 ° C.
    [0148] Cleaning the fragments.
    [0150] Subsequently, the fragment and the cut plasmid were purified using the commercial GeneJET Gel Extraction Kit (Thermo Scientific). For this, the following protocol was followed: a volume of Binding Buffer was added directly to the restriction mixture in relation to the volume of each reaction (1: 1 ratio, volume: volume), 100 ^ L in this case. The solution was then transferred to a GeneJET purification column, centrifuged for 1 min (12,000 rpm), and the resulting liquid was discarded. Subsequently, another 100 µL of Binding Buffer was added to the column, it was centrifuged for 1 min (12,000 rpm) and the resulting liquid was discarded. Then 700 µL of the Wash Buffer was added to the column, it was centrifuged for 1 min (12,000 rpm) and the resulting liquid was discarded. It was centrifuged again for 1 min (12,000 rpm) to eliminate the residues of the Wash Buffer. Elution was carried out with 40 µL of Elution Buffer, allowed to incubate for at least 1 min and centrifuged for 1 min (12,000 rpm). The cut and purified fragment and plasmid were visualized by means of a 1% agarose gel run for 1 h at 110 V and stained with Gel-Red.
    [0152] Ligation
    [0154] 3 ^ L of the vector (pET28a (+), 5369 bp) (12.8 ng / ^ l) was mixed with 10 ^ L of the UCA01 / 16.4 ng / ^ l fragment, respectively. It was incubated for 5 min at 70oC and then on ice for 15 min. Subsequently, 5 ^ L of 5X Rapid Ligation Buffer, 1 ^ L of the enzyme DNA ligase T4 (5U / ^ L) (Thermo Scientific) and 13 ^ L of nuclease-free water were added. Total reaction volume of 25 ^ L. Subsequently, it was incubated at 22oC for 1 hour 1 h.
    [0156] Transformation
    [0158] For the transformation procedure, competent cells were used E. coli Rosseta gami (DE3) and the following protocol was followed: 5 ^ L of the ligation mixture were added in 50 ^ L of competent cells, mixed with the pipet gently and incubated on ice for 20 min. Then a heat shock of 42 ° C was applied for exactly 45 sec, then immediately incubated on ice for 5 min. Subsequently, 200 µL of LB medium were added and it was incubated for 1 h at 37 ° C with shaking of 200 rpm. Solid medium plates (LB medium / kanamycin, 50 µg / ml) were then seeded with the reaction mixture and incubated overnight at 37 ° C.
    [0159] Isolation of plasmid DNA
    [0161] To carry out this procedure, the indications of the GeneJET Miniprep Kit (Thermo Scientific) commercial kit were followed according to the following protocol. 4.5 mL of the culture of the positive clones (LB / kanamycin medium, 50 ^ g / ml) were centrifuged (5000 rpm for 10 min) and the cell pellet was mixed with 250 ^ L of resuspension solution. 250 ^ L of lysis solution by vigorous mixing and inversion (4-6 times) until the solution became viscous and slightly clear. This procedure should not exceed 5 min in duration to avoid denaturation of the supercondensed plasmid DNA. Then 350 µL of neutralizing solution was added and mixed by inversion 4-6 times. Then it was centrifuged for 5 min (12,000 rpm). The supernatant was transferred to a GeneJET column being careful not to transfer the white precipitate. It was centrifuged for 1 min (12,000 rpm) and the resulting liquid was discarded. Subsequently, 500 µL of washing solution was added to the GeneJET column, it was centrifuged for 1 min (12,000 rpm) and the resulting liquid was discarded. The previous step was done in duplicate. It was centrifuged again for 1 min (12,000 rpm) in order to remove residues from the washing solution. The GeneJET column was then transferred to a clean 1.5 mL tube and 50 µL of elution buffer was added to the center of the column. It was incubated for 2 min at room temperature and centrifuged for 2 min (12,000 rpm).
    [0163] Colony PCR
    [0165] The reaction was carried out in the same way as for amplifying the respective fragments.
    [0167] Induction of expression
    [0169] An overnight culture of E. coli Rosseta gami (DE3) transformed with the vector pET28-Luci (complete gene) and with pET28, respectively, was used at a temperature of 37 ° C. Subsequently, fresh LB-kanamycin medium (50 ^ g / mL) (1:50 dilution) was inoculated. It was incubated at 37 ° C with shaking (250 rpm) until reaching an OD600 between 0.5-0.6. To induce protein expression, IPTG was added to a concentration of 1mM and incubated at 37 ° C with shaking (250 rpm) for 2.5-3 h. 1 mL of the induced culture was taken and mixed with 350 µL of resuspension buffer under native conditions (50 mM NaH2PO 4. -H2O, 300 mM NaCl, 10 mM imidazole, pH 8.0). It was frozen (liquid nitrogen) and thawed (37 oC) four times. It was then sonicated (60% amplitude) and centrifuged for 20 min at 8000 rpm. The soluble fraction was taken from the supernatant and the remaining pellet was resuspended in 350 ^ L of buffer of 10% run at 200 V for 45 min and stained with Coomasie blue (figure 3).
    [0171] Recombinant protein purification
    [0173] To carry out this procedure, the indications of the commercial His Spin Trap kit (GE Healtcare) were followed, which have an affinity for histidine, thus separating the recombinant protein thanks to the polyhistidine tail added at the N-terminal end, where 6 histidines were added to be able to carry out affinity purification, thus changing the natural sequence present in N. gaditana (Accession: XM_005854224.1). According to the purification protocol under denaturing conditions. For this, a volume of 50 mL ( E. coli Rosseta gami (DE3)) was cultured, which were induced as explained previously. Subsequently, it was centrifuged at 10,000 rpm for 10 min in order to collect the cell pellet. Then 1 mL of binding buffer was added to resuspend the pellet obtained from 50 mL of culture. Subsequently, a mechanical lysis was performed using a sonicator (60% amplitude in 5-second intervals on ice). In order to clarify the lysate, it was centrifuged at 15,000 g for 10 min to collect the supernatant. Then, each His Spin Trap column was inverted and repeatedly shaken in order to resuspend their storage medium. The lid was opened a quarter turn and the bottom seal was removed. The column was placed in a clean 2 mL tube and centrifuged for 30 sec at 100 g, after which the cap was discarded. To equilibrate the column, 600 µL of binding buffer was added and it was centrifuged for 30 sec at 100 g. Next, up to 600 µL of the sample was added at a time, centrifuging for 30 sec at 100 g each time. The column can be used several times as long as its capacity is not exceeded (600 ^ L). For washing, 600 ^ L of the binding buffer were added and it was centrifuged for 30 sec at 100 g. This procedure was performed in duplicate). Finally, it was eluted by adding 200 µL of the elution buffer (500 mM imidazole) in duplicate and centrifuging for 30 sec at 100 g. The first 200 ^ L of elution contain most of the purified protein (Figure 4). This purification could be carried out due to the polyhistidine tail bound to the protein, provided by the plasmid pET28a.
    [0175] Biological Activity
    [0177] The lyophilized samples were initially dissolved with dimethylsulfoxide (DMSO) at pH = 2, and later, for their application to cell cultures, they were diluted in DMEM culture medium (ATCC) supplemented with 100 U / mL penicillin and 100 ^ g / mL streptomycin. (PAN Biotech), until a final DMSO concentration of less than 0.5% is obtained. 6 samples were purified with a content between 1.2-2.74 ^ g of protein (total = 10.46 ^ g of protein).
    [0178] All cell lines were obtained from the American ATCC ( American Type Culture Collection) certified collection and the following were used:
    [0179] • the human colorectal adenocarcinoma epithelial cell line: Caco-2 (ATCC® HTB-37).
    [0180] • the human hepatocellular carcinoma cell line: HepG2 (ATCC®-HB-8065).
    [0181] • the human endothelial cell line EA.hy926 (ATCC® CRL-2922 ™).
    [0183] The cell lines were grown and kept at 37 ° C and 5% CO2 in the medium recommended by the ATCC. For the Caco-2 cell line the EMEM medium was used and for the HepG2 and EA.hy926 cell lines the DMEM medium. These media were supplemented with 10% fetal bovine serum (FBS), and 100 U / mL of penicillin and 100 µg / ml of streptomycin.
    [0185] Evaluation of cell viability to evaluate the antiproliferative effect
    [0187] The evaluation of the antiproliferative activity in the cells in contact with prohibitin was carried out in the human cell lines Caco-2, HepG2 and EA.hy926. For this, the cells were seeded in 96-well plates and incubated with a range between 0.7 and 0.003 ^ g / mL of prohibitin at 37 ° C and 5% CO2 at different contact times. The contact time in the two tumor lines HepG2 and Caco-2 was 24 h, while in the non-tumor cell line EAhy.926 the contact times were 6 h and 72 h. After the incubation period, the cell viability was evaluated by means of a fluori-colorimetric assay with the alamarBlue® reagent (invitrogen). Fluorescence was measured at 540nm / 590nm Excitation / Emission by a Fluostar Optima plate spectrofluorimeter (BMG Labtechnologies). Given the direct relationship between fluorescence units and cell viability, the viability calculation was performed with respect to the control cells with the equivalent amount of DMSO in the samples, using the following formula:
    [0189] % Viability = (Sample Fluorescence Units / Control Fluorescence Units) x 100
    [0191] In the graphs of figure 5 it is observed that the purified recombinant prohibitin protein showed antiproliferative capacity on HepG2 and Caco-2 cancer cells when a significant reduction in proliferation was observed after treatment with prohibitin. In the case of Caco-2, a dose-dependent effect was observed, so that the higher the concentration of prohibitin, the greater the antiproliferative effect. In the case of the HepG2 line, a dose-dependent effect was not observed. The application of prohibitin on the non-tumor line Eahy.926 to two prohibitin has a selective effect and only acts on tumor cells.
    [0193] References
    [0195] Fernández-Acero, FJ, Amil-Ruiz, F., Durán-Peña, MJ, Carrasco, R., Fajardo, C., Guarnizo, P., Vallejo, RA (2018). Valorization of the microalgae Nannochloropsis gaditana biomass by proteomic approach in the context of circular economy. Journal of Proteomics. https://doi.org/10.1016/JJPROT.2018.10.015.
    [0196] Mishra, S., Murphy, LC, & Murphy, LJ (2006). The Prohibitins: emerging roles in diverse functions. Journal of Cellular and Molecular Medicine, 10 (2), 353-363. https://doi.org/10.1111/j.1582-4934.2006.tb00404.x.
    [0197] Theiss, AL, & Sitaraman, SV (2011). The role and therapeutic potential of prohibitin in disease Theiss, AL, & Sitaraman, SV (2011). The role and therapeutic potential of prohibitin in disease. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1813 (6), 1137 1143. https: // do. Biochimica et Biophysica Acta ( BBA) - Molecular Cell Research, 1813 (6), 1137-1143. https://doi.org/10.1016J.BBAMCR.2011.01.033.
    [0198] Thrush, SF, Hewitt, JE, Gibbs, M., Lundquist, C., & Norkko, A. (2006). Functional Role of Large Organisms in Intertidal Communities: Community Effects and Ecosystem Function. Ecosystems, 9 (6), 1029-1040. https://doi.org/10.1007/s10021-005-0068-8.
    [0199] V.thurman. (2007). Studyguide for Introductory Oceanography by Thurman.
    [0200] Van Aken, O., Pecenková, T., van de Cotte, B., De Rycke, R., Eeckhout, D., Fromm, H., Van Breusegem, F. (2007). Mitochondrial type-I prohibitins of Arabidopsis thaliana are required for supporting proficient meristem development. The Plant Journal, 52 (5), 850-864. https://doi.org/10.1111/j.1365-313X.2007.03276.x
    [0201] http://bioinfo.ut.ee/prímer3-0.4.0/
    权利要求:
    Claims (13)
    [1]
    1. - A recombinant protein obtainable by a procedure that comprises:
    a) integrate an insert with the nucleotide sequence SEQ ID NO. 1 in an expression vector,
    b) transforming a host with the expression vector from step (a), c) inducing the expression of the recombinant protein,
    d) extracting the recombinant protein, and
    e) purifying the recombinant protein.
    [2]
    2. - The recombinant protein according to the preceding claim, wherein the host of step (b) are expressing bacteria.
    [3]
    3. - The recombinant protein according to the preceding claim, wherein the expression bacteria are E. coli BL21 (DE3) bacteria.
    [4]
    4. - The recombinant protein according to any of claims 1-3, wherein the integration of step (a) is carried out through a ligation process.
    [5]
    5. - The recombinant protein according to any of claims 1-4, wherein the insert was synthesized using the nucleotide sequence primers SEQ ID NO: 3 and SEQ ID NO: 4.
    [6]
    6. - A composition comprising:
    a) a protein consisting of the amino acid sequence SEQ ID NO: 2, or b) the recombinant protein obtainable according to any of claims 1-5.
    [7]
    7. The composition according to the preceding claim, which is a pharmaceutical composition.
    [8]
    8. - A composition according to any of claims 6 or 7, for use in therapy.
    [9]
    - A composition according to any of claims 6 or 7, for use in the prevention or treatment of cancer, cell proliferation disorders or unwanted angiogenesis, or to treat or prevent a solid tumor in a subject, such and such as a colorectal, esophagus, lung, prostate, breast, pancreas, or liver tumor or cancer.
    [10]
    10. The composition for use according to claim 9, wherein said use is in the prevention or treatment of a solid tumor in a subject.
    [11]
    11. - The composition for use according to claim 9, wherein said use is in the prevention or treatment of a solid tumor selected from the list consisting of a tumor or colorectal cancer, esophagus, lung, prostate, breast , pancreas or liver.
    [12]
    12. - The composition for use according to claim 9, wherein said use is in the prevention of the development of cancer associated with chronic inflammation.
    [13]
    The composition for use according to claim 9, wherein said use is in the prevention of the development of cancer associated with chronic inflammation caused by Crohn's disease, ulcerative colitis, pancreatitis, or cirrhosis.
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US20130158237A1|2011-12-16|2013-06-20|Panasonic Corporation|Recombinant protein capable of binding specifically and quickly to troponin i derived from human myocardium|
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