ES2810229B2 - RECOMBINANT PROTEIN OF NANNOCHLOROPSIS GADITANA - Google Patents

Description

DESCRIPTION

RECOMBINANT PROTEIN OF NANNOCHLOROPSIS GADITANA

TECHNICAL SECTOR

The invention belongs to the field of biomedicine, in particular to the use of a recombinant protein from N. gaditana , also known as Microchloropsis gaditana (Fawley, Jameson, & Fawley, 2015), which, when purified, has an anti-proliferative effect on tumor cells. .

BACKGROUND OF THE INVENTION

Cancer therapy has experienced important advances that have occurred with unusual speed. The therapeutic arsenal has intensified in efficacy with cytostatics or what is commonly known as "chemotherapy", drugs with different mechanisms of action that induce apoptosis or cell death but with 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 for intravenous administration, however in recent years we have witnessed a real "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 in a very important way to an improvement in the control of the disease or in the quality of life of patients.

Even so, there is still a need to provide new therapies that induce apoptosis or cell death or prevent tumor growth, and that have a high specificity against tumor cells.

BRIEF DESCRIPTION OF THE INVENTION

The present invention refers to the use of a recombinant protein as an inhibitor of tumor growth, specifically to the use of the recombinant UCA01 protein, which is a protein that submerges 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, increased p53 protein activity, and protection from mitochondria degradation. Specifically, in the present invention, the biological activity of the recombinant UCA01 protein extracted from microalgae ( 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 a "hypothetical protein" in the NCBI (Accession: XM_005854224.1).

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 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 follows that the recombinant UCA01 protein from N. gaditana of the invention has a selective anti-proliferative (cytotoxic) effect since it only acts on the tumor cells tested.

Finally, it is important to highlight, by virtue of what has been said above, 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 assays of biological activity in other proteins, which indicates a new method and a new source for obtaining UCA01, efficient, since the quantities necessary to carry out its antitumor activity have been minimal.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Functional domains of prohibitin 1 (Theiss & Sitaraman, 2011).

Figure 2: (a) Primers designed for the amplification of the complementary DNA of N. gaditana , with the restriction enzymes used to create cohesive ends with the plasmid pET28a; (b) Agarose gel amplification confirmation; (c) Blast homology sequencing confirmation of 100% with UCA 01 homologues; (d) Matrix of percent identity, created by Clustal 2. 1, first, UCA01 protein, compared NCBI sequencing: with N. gaditana prohibitin and several human prohibitins.

Figure 3: 10% SDS-PAGE gel; lines 3, 5 and 7 expression of the UCA01 of N. gaditana by the bacterium E. coli, Rosseta gami (DE3).

1: 0.4 mM IPTG

2: Not induced

3: 0.6 mM IPTG

4: There is no sample.

5: 0.8 mM IPTG

6: Not induced

7:1 mM of IPTG

8: Not induced

Figure 4: Image of the SDS-PAGE gel at 10% of the UCA01 purification process.

M.: Molecular weight marker (Kda)

A: Not induced without purification

B: Induced without purification

C: First pass of sample induced by affinity column

D: Wash step

E: Elution 1

F: Elution 2

Inside the box we will find the band belonging to the UCA01 protein

Figure 5: Antiproliferative effect of the recombinant protein UCA01 from N. gaditana. The effect on the 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. Statistics used: t-student comparing each value with the control with DMSO.

DESCRIPTION OF THE INVENTION

Definitions

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.

The term "inhibit proliferation" refers to any method that decreases the ability of cells to grow.

"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 unicellular eukaryotes. Prohibitins are divided into two classes, designated Type I and Type II prohibitins, based on their similarity to the yeast PHB1 and PHB2 genes, respectively. Every organism has at least one copy of each type of prohibitin gene (Mishra, Murphy, & Murphy, 2006; Van Aken et al., 2007).

The term “mycmalga” refers to microscopic algae, generally found in freshwater and marine systems, that live 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 range 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 greenhouse gas carbon dioxide to grow photoautotrophically. Microalgae, together with bacteria, form the base of the food web and provide energy for all trophic levels above them (Thrush et al., 2006).

DESCRIPTION

RECOMBINANT PROTEIN OF THE INVENTION.

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 the microalgae ( N. gaditana), classified until 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 only to 56.36% (clustal W) with the homologous human prohibitin (figure 2), which, a priori, would not allow us to deduce that it had the same functions as human prohibitin. In fact, when compared to several human prohibitins (GenBank: S85655.1, GenBank: L14272.1, GenBank: BC095460.1, GenBank: BT007411.1), the homology found was 56.34% and 56.22%, respectively. %, still small percentages that, again, did not allow us 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 microalga Nannochlompsis Cadiz Lubian CMP 572 (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:

SEQ ID NO 1:

ATGTCTCCAGCAGGACCGCTGGGAGCCTTGCTAGGTGTGACGGGCATCCTTTACGCCG GGTACAATAGTTTTTACACGGTGGAGGGTGGGCACCGAGCTCTGTTGTTCAACAGATTA ATCGGTGTGAAAGAAGAAGTGTACATGGAGGGAATGCATTTTATGATTCCCTGGTTCGA CATGCCCATCATTTACGACATCCGCCCCAAGCCCCGGATGATCCAGTCCTTGACAGGAA GCAAAGACATGCAAATGGTCAACATCACCATCCGCGTTTTGTCTAAGCCCGACTCGGCT CAACTCCGCTGGATCTTCCGCACCTTGGGTCGCGACTACGACGAGCGTGTCCTCCCTT CCATCGTCAACGAGGTCTCCAAGGCCGTGGTGGCCAAGTACAACGCCGCGGAGCTCTT GACGAAGCGTGAGATGGTCTCCACCCAAATCCGGTTGCAGTTGGAGAAGCGTGCGAAG GAGTTTCGGATCGTCCTGGACGACGTGTCGATCACCCACTTGACCTTCTCCCGGGAGT ACACGAACGCGGTCGAGGCCAAGCAAGTTGCTCAACAGGAAGCCGAGCGCGCGAAAT ATGTGGTAATGAAAGCGAACCAAGAAAAGGAAGCCATCATTATCAAAGCGGAGGGAGA GGCCCAATCCGCTGCTCTGGTGGGTAAGGCCATTCGAGAGAATCCTGCTTTTATCAAGC TGCGCAAAATCGACGCAGCCAGGGACATTGCGAATGTCGTGTCCTCTTCGGGTCAGAA GGTCTATCTGTCTGCGGACTCCCTCTTGTTGAATATGTACTCGGGCGACGAAGAGAAGT CTGGAAAGAAGCGGTAGGCGGCCGCACTCGAGCACCACCACCACCACCAC,

from which the following amino acid sequence was obtained (SEQ ID NO 2):

MSPAGPLGALLGVTGILYAGYNSFYTVEGGHRALLFNRLIGVKEEVYMEGMHFMIPWFDMPI IYDIRPKPRMIQSLTGSKDMQMVNITIRVLSKPDSAQLRWIFRTLGRDYDERVLPSIVNEVSK AVVAKYNAAELLTKREMVSTQIRLQLEKRAKEFRIVLDDVSITHLTFSREYTNAVEAKQVAQQ EAERAKYWMKANQEKEAINKAEGEAQSAALVGKAIRENPAFIKLRKIDAARDIANWSSSGQ KVYLSADSLLLNMYSGDEEKSGKKRAAALEHHHHHH,

it was synthesized from the microalgae Nannochloropsis gaditana Lubian CMP 572 (Fernández-Acero et al., 2018). For this, once the proteome of this microalgae 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 Ndel and SalI cutting enzymes, thus joining the plasmid together with the complementary DNA, introducing it into the Top10 strain of E. coli, used as a storage strain of E. coli. the construction.

Once it was confirmed by PCR in the Top10 strain of E. coli, 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 E.coli rosetta gami , to be used as expression strain, (DE3) UCA01 protein was expressed using IPTG as expression induction method and confirmed on an acrylamide SDS-page gel (Figure 3).

Following production of the recombinant protein in the host cell, the cell lysate was passed over an affinity column for purification. The tagged fusion protein 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 histidine affinity columns (GE Healthcare), thanks to the N-terminal polyhistidine tag (6 histidines) present in the Pet-28a plasmid and added to the protein.

From said cloning and purification, the authors of the present invention demonstrated, for the first time, the antitumor activity of this recombinant protein from N. gaditana in the HepG2 hepatic tumor line and the Caco-2 colon cancer tumor line, demonstrating that inhibited proliferation as well as cell viability of these tumor lines. In addition, this effect maintains a directly proportional correlation to the concentration of recombinant UCA01, where a higher concentration of this, greater inhibition, or what is the same, lower cell viability of the tumor line.

Therefore, a first aspect of the invention relates to a recombinant protein obtainable by a process comprising:

a) integrating an insert with a nucleotide sequence comprising or consisting of SEQ ID NO. 1, in a genetic construct or an expression vector,

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,

c) transforming a host with the genetic construct or with the expression vector of step (a) or step (b), where said host is an expression inducing strain,

d) inducing the expression of the recombinant protein,

e) extracting the recombinant protein, and

f) optionally purifying the recombinant protein

It is noted that step a) above can, alternatively, be carried out by inserting a variant nucleotide sequence that has an identity of at least 80% with SEQ ID NO: 1.

The term "identity" as used herein refers to the proportion of identical amino acids or nucleotides between two amino acid or nucleotide sequences being compared.

The term "homology" as used herein refers to the similarity between two structures due to common evolutionary ancestry, and more specifically, to the similarity between the amino acids of two or more proteins or amino acid sequences.

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 of similarity or identity greater than 30% indicate homologous structures. We can therefore consider that identity percentages of at least 80% will include the conserved active regions.

In a more preferred embodiment of this aspect of the invention, the nucleotide sequence of step a) will have 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 have an identity of at least 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: 1.

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 5' or 3' to the cDNA or when they are part of an intron. Regulatory sequences include, in addition to promoters, translation facilitating sequences, processing signals for introns, stop codons, signal sequences, internal ribosome binding sites (IRES), and polyadenylation signals.

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. Those 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.

In a preferred embodiment of this aspect of the invention, the expression vector is the prelinearized vector pET28-Luci.

A second aspect of the invention refers to host cells or organisms such as expression-inducing strains that comprise the expression vector or the gene construct, as these have been defined in the invention. In principle, any type of host organism known to those 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 (dicots or monocots), an insect cell, eg, baculovirus, a mammalian cell (COS, CHO, C127, HeLa cells, and the like), and a non-human transgenic (eg, example, a mouse, a cow, a goat, a rabbit, a pig, etc.).

In another preferred embodiment of this aspect of the invention, the expression bacteria are chemically competent Escherichia coli cells, which have a suitable genotype. for transformation and protein expression, and whose genome is known (Genome sequences of Eschenchia coli B strains REL606 and BL21(DE3). Jeong H, et al. J Mol Biol 2009 Dec 11).

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 memory, the expression bacteria are those that possess the necessary machinery to overexpress the inserted cDNA and produce the recombinant protein.

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.

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 union of the insert to the plasmid thanks to the homology in the nucleotide sequence present in both.

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 joining single-stranded DNA molecules that have short, homologous sequences at their ends, such as an amplified PCR product and a vector.

In another preferred embodiment, the insert of step a) of the first aspect of the invention is synthesized using the primers of nucleotide sequence SEQ ID NO: 3 and SEQ ID NO: 4.

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 its 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 drug.

It is noted that the third aspect of the invention does not only refer to the amino acid sequence that comprises or consists of SEQ ID NO: 2, but also to 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").

A fourth aspect of the invention relates 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.

A fifth aspect of the invention is 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 colorectal, esophageal, lung, prostate, breast, pancreas and liver tumor or cancer, 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 decrease or block tumor cell growth. In one embodiment of the invention, the composition can be administered to patients with solid tumors, such as colorectal, esophageal, lung, prostate, breast, pancreas, and liver tumor or cancer, by blocking cancer cell proliferation, even in presence of inflammatory stimuli.

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 inhibiting the development of tumors associated with inflammation and their metastases, which comprises the administration of the recombinant protein to a subject that needs it. 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.

Likewise, the administration of the recombinant protein of SEQ ID NO 2 can be used, 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.

Regarding the dose and treatment schedule to follow with the compositions that comprise the recombinant protein of SEQ ID NO 2 or variants thereof, a person skilled in the art can easily determine the dose and treatment schedule (prophylactic or therapeutic). Effective amounts may vary depending on the relative potency of the individual compositions, and may be calculated based on the molecular weight of the recombinant protein in vitro or on animal studies. For example, given the molecular weight of the compound (chemical structure) and an experimental effective 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.

On the other hand, a sixth aspect of the invention refers 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 vehicles.

The invention also provides cancer diagnostic compounds 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 nonfluorescent group, a fluorescent semiconductor particle, a paramagnetic or superparamagnetic agent, and a radioisotope.

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 treatment agent by covalent bonds. In other cases, the protein is conjugated to the treating agent by a linker element.

The invention also includes 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 chemotherapy or radiotherapy treatment. The elements that form these combinations can be administered, to individuals in need, in the course of medical treatment, sequentially or simultaneously. In an embodiment of the invention, said standard chemotherapy-specific drug 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.

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 therapeutic effectiveness.

The following examples serve to illustrate the present invention but are not intended to limit it.

EXAMPLES

Experimental development.

Once the amplification of this protein was verified, the recombinant protein was made, following the following steps.

Isolation of total RNA from N. gaditana

Total RNA was isolated using NucleoSpin RNA® (Macherey-Nagel) following the manufacturer's instructions. In short. The quality of the isolated RNA was verified, following the indications of the Agilent RNA 6000 Nano© Kit.

Synthesis of complementary DNA by RT-PCR

To carry out this procedure, the instructions of the qScript® commercial kit (Quanta Biosciences) were followed. The reaction protocol used was as follows: 1 ^L of the isolated RNA (1-10 ^g of total RNA), 14 ^L of nuclease-free water, 4 ^L of the mixture of qScript reaction (5X) and 1 jL of qScript reverse transcriptase; for a final volume of 20 jL. It was gently vortex mixed and then centrifuged for 10 sec. Subsequently, they were placed in a thermocycler under the following program: 1 cycle at 22°C for 5 min., 1 cycle at 42°C for 30 min., and one cycle at 85°C for 5 min. with a stop at 4°C.

PCR amplification

For this procedure, the indications of the Phusion Flash High-Fidelity PCR Master Mix commercial kit (Thermo Scientific) were followed. The reaction conditions were as follows: 25 jL of 2X Phusion Flash PCR Master Mix, 2.5 jL of primer 10 jM Prohi-F: 5'-GGCATATGTCTCCAGCAGGACCGCTGG-3' (cut site for Ndel) (SEQ ID NO 3 ), 2.5 |jL of the first 10 |jM Prohi-R: 5'-GGGTCGACCTACCGCTTCTTTCCAGACTTC-3' (Salí cut site) (SEQ ID NO 4), 2 jL of cDNA (65 ng/jL) and 18 jL of water, for a total reaction volume of 50 jL. With the amplification with the Prohi-F and Prohi-R primers, an 834 bp fragment is obtained (GenBank XM_005854224.1). 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 using a 1% agarose gel run for 1 h at 110 V and stained with Gel-Red (figure 2).

Purification of the amplified fragment.

This procedure was carried out following the instructions of the commercial GeneJET Gel Extraction Kit (Thermo Scientific). For this, the following protocol was followed: once the band was cut, it was weighed and a volume of Binding Buffer was added in relation to the weight of the band (ratio 1:1, 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 jL of Wash Buffer were 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 Wash Buffer residues. Elution was performed with 40 jL of Elution Buffer, incubated for at least 1 min and centrifuged for 1 min (12,000 rpm). The fragment purified from the gel bands was visualized on a 1% agarose gel run for 1 h at 110 V and stained with Gel-Red.

Digestion with restriction enzymes.

In order to linearize the pET28a vector (Novagen) to ligate 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 Ndel (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 μl of FastAp 10X buffer, 2 μl of alkaline phosphatase (1 U/ ^l) were added and incubated for 1 h at 37°C. With the UCA01 fragment, the following reaction was carried out: 20 jL of the amplified and purified DNA fragment (31.1 ng/|jL), 10 | ^j L of 10X Buffer D, 2 | ^j L of Ndeí (10U/jiL), 2 | ^j L of Salí (10U/jiL) and 66 | ^j L of water. Total reaction volume of 100 jL. It was incubated for 4 h at 37°C.

Cleaning of the fragments.

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 jL 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 jL of Binding Buffer were added to the column, it was centrifuged for 1 min (12,000 rpm) and the resulting liquid was discarded. Then 700 jL of Wash Buffer were 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 Wash Buffer residues. Elution was performed with 40 jL 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 on a 1% agarose gel run for 1 h at 110 V and stained with Gel-Red.

ligation

3 jL of the vector (pET28a (+), 5369 bp) (12.8 ng/^l) were mixed with 10 jL of the UCA01 fragment /16.4 ng/^l), respectively. It was incubated for 5 min at 70oC and then on ice for 15 min. Subsequently, 5 jL of 5X Rapid Ligation Buffer, 1 jL of the enzyme DNA ligase T4 (5U/jL) (Thermo Scientific) and 13 jL of nuclease-free water were added. Total reaction volume of 25 jL. Subsequently, it was incubated at 22oC for 1 hour.

Transformation

For the transformation procedure, competent E. coli Rosseta gami (DE3) cells were used and the following protocol was followed: 5 ^L of the ligation mixture were added to 50 ^L of the competent cells, mixed gently with the pipette and incubated on ice for 20 min. A heat shock of 42°C was then applied for exactly 45 sec, followed by immediate incubation on ice for 5 min. Subsequently, 200 ^L of LB medium were added and incubated for 1 h at 37°C with 200 rpm agitation. Plates of solid medium (LB medium/kanamycin, 50 µg/ml) were then seeded with the reaction mixture and incubated overnight at 37°C.

Plasmid DNA isolation

To carry out this procedure, the indications of the commercial GeneJET Miniprep Kit (Thermo Scientific) 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 mixing vigorously and by inversion (4-6 times) until the solution becomes viscous and slightly clear. This procedure should not exceed 5 min in duration to avoid denaturation of the supercondensed plasmid DNA. Then 350 ^L of neutralizing solution was added and mixed by inversion 4-6 times. It was then 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 ^L of washing solution were 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 eliminate residues of the washing solution. The GeneJET column was then transferred to a clean 1.5 mL tube and 50 ^L 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).

ColonyPCR

The reaction was carried out in the same way as for amplifying the respective fragments.

Induction of expression

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 OD ⁶⁰⁰ between 0.5-0.6. To induce protein expression, IPTG was added up to a concentration of 1 mM 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 ^L of resuspension buffer under native conditions (50 mM NaH ² PO ⁴ --H ² O, 300 mM NaCl, 10 mM imidazole, pH 8.0). It was frozen (liquid nitrogen) and thawed (37 °C) four times. It was then sonicated (60% amplitude) and centrifuged for 20 min at 8,000 rpm. The soluble fraction was taken from the supernatant and the remaining pellet was resuspended in 350 ^L of solubilization buffer under native conditions. The result was visualized by means of SDS-PAGE at 10% run at 200 V for 45 min and stained with Coomasie blue (figure 3).

Recombinant protein purification

To carry out this procedure, the indications of the commercial kit His Spin Trap (GE Healthcare) 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 carry out the 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 was cultured ( E. coli Rosseta gami (DE3)), which were induced as explained above. 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, mechanical lysis was performed using a sonicator (60% amplitude at 5-sec 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 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 lid was discarded. To balance the column, 600 ^L of binding buffer were added and it was centrifuged for 30 seconds at 100 g. Next, up to 600 ^L of the sample were added at a time, centrifuging for 30 seconds 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 binding buffer were added and centrifuged for 30 seconds at 100 g. This procedure was performed in duplicate). Finally, eluted adding in duplicate, 200 ^L of the elution buffer (500 mM of imidazole) and centrifuging for 30 sec at 100 g. The first 200 ^L of elution contains most of the purified protein (figure 4). This purification could be carried out due to the polyhistidine tail attached to the protein, contributed by the plasmid pET28a.

Biological Activity

The lyophilized samples were initially dissolved with dimethyl sulfoxide (DMSO) at pH=2, and later, for 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 obtaining a final concentration of DMSO less than 0.5%. 6 samples with a content between 1.2-2.74 ^g of protein (total = 10.46 ^g of protein) were purified.

cell lines

All cell lines were obtained from the American certified collection ATCC ( American Type Culture Collection) and the following were used:

• the human colorectal adenocarcinoma epithelial cell line: Caco-2 (ATCC® HTB-37).

• the human hepatocellular carcinoma cell line: HepG2 (ATCC®-HB-8065). • the human endothelial cell line EA.hy926 (ATCC® CRL-2922™).

Cell lines were grown and maintained at 37°C and 5% CO ² in the medium recommended by the ATCC. For the Caco-2 cell line, EMEM medium was used and for HepG2 and EA.hy926 cell lines, DMEM medium. These media were supplemented with 10% fetal bovine serum (FBS), and 100 U/mL penicillin and 100 µg/mL streptomycin.

Evaluation of cell viability to evaluate the antiproliferative effect

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% CO ² 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, cell viability was evaluated using a fluori-colorimetric assay with the alamarBlue® reagent (invitrogen). Fluorescence was measured at Aexcitation/Aemission of 540 nm/590 nm using a Fluostar Optima plate spectrofluorometer (BMG Labtechnologies). Given the direct relationship between fluorescence units and cell viability, viability was calculated with respect to control cells with the equivalent amount of DMSO in the samples, using the following formula:

% Viability = (Fluorescence Units sample/ Fluorescence Units control) x 100

The graphs in Figure 5 show that the purified recombinant prohibitin protein showed antiproliferative capacity on HepG2 and Caco-2 cancer cells, as 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, no dose-dependent effect was observed. The application of prohibitin on the non-tumor line Eahy.926 at two times (6 and 72 h) did not reduce cell viability. Therefore, a priori it seems that prohibitin has a selective effect and only acts on tumor cells.

References

Fernández-Acero, FJ, Amil-Ruiz, F., Durán-Peña, MJ, Carrasco, R., Fajardo, C., Guarnizo, P., Vallejo, RA (2018). Valorisation 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.

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.

Theiss, A.L., & Sitaraman, S.V. (2011). The role and therapeutic potential of prohibitin in disease. Molecular Cell Research, 1813(6), 1137-1143. https://doi.org/10.1016J.BBAMCR.2011.01.033.

Thrush, SF, Hewitt, J.E., 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.

V.thurman. (2007). Study guide for Introductory Oceanography by Thurman.

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

http://bioinfo.ut.ee/primer3-0.4.0/

Claims (3)

1. - A pharmaceutical composition comprising a protein consisting of the amino acid sequence SEQ ID NO: 2. 2. - A pharmaceutical composition according to the preceding claim, for use in therapy. 3. - A pharmaceutical composition according to claim 1, for use in the prevention or treatment of colorectal or liver cancer.