RU2647771C2 - Method for obtaining recombinant human orexin a , plasmid dna, strain producer - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention refers to the recombinant production of human proteins and can be used in order to prepare human orexin A in the Escherichia coli cells. Production method consists in culturing the cells of the producer strain Escherichia coli BL21(DE3)pET151/D-TOPO, which is obtained by means of transformation of plasmid DNA pET151/D-TOPO, which contains the codon optimized human orexin A gene, of the Escherichia coli cells BL21 (DE3) using the inducer of the target protein expression. Cells are then lysed, the inclusion bodies are washed with 0.2 M sodium deoxycholate, the inclusion bodies are dissolved in the 8 M urea solution, orexin A is refolded in 0.1M Tris pH 8.0 buffer, 0.2 mM EDTA with 0.5 M L-arginine and chromatographic purification of the resulting protein solution on the S-Sepharose column and on the S-100 column is carried out.

EFFECT: invention allows to obtain the highly purified physiologically active recombinant human orexin A.

3 cl, 3 dwg, 2 tbl, 5 ex

Description

A group of inventions related to biotechnology, pharmacy and medicine is proposed. The invention can be applied to obtain human orexin A.

Orexins, or hypocretins, are neuropeptides discovered in 1998 independently by two research groups (T. Sakurai et al., 1998, de Lecea L. et al., 1998). Orexins A and B are translated from one mRNA in the form of a preproorexin precursor peptide consisting of 130 amino acid residues (Peyron C., et al., 1998). The human gene encoding the preproorexin protein is located on the 17th chromosome and has 2 exons that are fully translated into the final peptides. Preproorexin has an N-terminal signal sequence of 33 amino acids. After removal of this sequence, proorexin is formed, from which prohormone convertase cuts out two amino acid sequences with the formation of orexins A and B (Sakurai T. et al., 1999).

Mammalian orexin A is an oligopeptide consisting of 33 amino acids, and its approximate mass is 3.5 kDa. The orexin A molecule has a loop-like spatial structure amidated at the C-terminus; its N-terminus contains a pyroglutamyl residue and two intramolecular disulfide bonds: Cys6-Cys12 and Cys7-Cys14 (Sakurai T. et al., 1998).

Orexins are synthesized by a small population of hypothalamic neurons (about 1100-3400 in rats, 50000-80000 in humans) (Harrison, T.A., et al., 1999; Peyron C., et al., 1998; Thannickal T.C.et al., 2000), the most abundant in the periferic zone of the lateral hypothalamic region (De Lecea L. et al., 1998; Sakurai T. et al., 1998). The processes of orexin-containing cells are projected into various areas of the brain and spinal cord, for example, into the thalamus, components of the limbic system (hippocampus, amygdalla), pineal gland, cerebral cortex, spinal cord (I and X plates) (Date Y., et al., 2000; Date Y., et al 2000; Peyron C. et al., 1998).

Orexin receptors and processes of orexin-containing neurons are found in many nuclei at various levels of the central nervous system involved in the regulation of the sleep / wake cycle (blue spot, bridge, reticular formation, suture nuclei, mammary nuclei, lateral preoptic region of the hypothalamus) (Kukkonen JP et al ., 2002). The activity of orexin-containing neurons is subject to daily fluctuations, it increases during activity and decreases during dormancy (Estabrooke I.V. et al., 2001; Yoshida Y et al., 2001). Normally, the amount of orexin rises in the morning, leading to awakening (Taheri, 2001).

The main action of orexin is to initiate awakening and maintain an active state, mainly due to the activation of wakefulness centers. Injections of orexin A into the structures involved in the regulation of the sleep / wake cycle provoke wakefulness in rats and cats, and also inhibit both phases of sleep (Bourgin P. et al., 2000; Espana RA et al., 2001; Hagan JJ et al ., 1999; Huang ZL et al., 2001; Methippara MM et al., 2000; Piper DC et al., 2000).

Orexin-containing neurons are involved in the regulation of nutritional behavior, acting as a trigger that triggers search nutritional behavior. Intraventricular administration of orexin A initiates food intake in rats (Sakurai T. et al., 1998). The introduction of the first type of orexin receptor antagonist, SB-334867, suppresses the basic level of eating behavior, normal weight gain, and the appetite-stimulating effect caused by microinjections of orexin A into the hypothalamus (Haynes A.S. et al., 2000).

Outside the central nervous system, orexin-containing neurons are found in the duodenum, stomach, pancreas, adrenal glands, heart, testes, as well as in the mesenteric sympathetic ganglia (Arihara, Z., et al., 2000; Johren, O., et al 2001; Karteris, E, et al., 2001 Kirchgessner, AL, et al 1999; Naslund, E., et al., 2002; Randeva HS et al., 2001).

Orexins affect the functions of the endocrine system and through the regulation of the synthesis and secretion of various hormones affect the autonomic functions of the body. Analysis of modern literature indicates the involvement of orexin-containing neurons in thermoregulation, which consists in maintaining circadian changes in body temperature, as well as modulating them in various forms of pathology (Mochizuki Takatoshi, Elizabeth V. Klerman, Takeshi Sakurai, Thomas E. Scammell. Elevated body temperature during sleep in orexin knockout mice. Am. J. Physiol. Regul. Integrative Comp.Physiol. 291: 533-540. 2006, Szekely M., Petervari E., Balasko M., Hernadi I., Uzsoki B. Effects of orexins on energy balance and thermoregulation. Regul. Pept. 104 (1-3): 47-53. 2002, Tanaka M., Tonouchi M., Hosono T., Nagashima K., Yanase-Fujiwara M., Kanosue K. Hypothalamic region facilitating shivering in rats. J.pn. J. Physiol. 51: 625-629. 2001).

The complex of data obtained suggests the involvement of the orexin-containing neuron system in the formation of the systemic response, in particular, the components of the prodromal syndrome, to antigen administration and in the mechanisms of central regulation of the immune system functions.

Increased secretion of major stress neurotransmitters and hormones caused by the action of orexin, suggests the possible participation of orexin-containing neurons in the mechanisms of regulation of stress-induced reactions of the body. These data are consistent with studies describing animal behavioral responses to intraventricular administration of orexin A. For example, grooming and defensive behavior caused by this exposure are typical of a generalized stress response (Duxon M.S. et al., 2001; Espana RA et al., 2001; Ida T. et al., 1999; Ida T. et al., 2000).

The role of orexin in the pathogenesis of diseases such as Huntington's chorea (Petersen A. et al., 2005), Parkinson's disease (Yasuia K. et al., 2006), obstructive sleep apnea syndrome (Sakurai S. et al., 2005), type II diabetes (Goncz E. et al., 2007, Cai J. et al., 2006). In some forms of severe traumatic brain injury, a decrease in orexin concentrations and atrophy of orexin-containing neurons in patients is detected (Baumann C.R. et al., 2005).

Orexins improve cognitive functions such as attention and memory. It has been shown that intranasal or intravenous administration of orexin A after forced wakefulness for 30-36 hours significantly improves the test results for short-term memory in rhesus monkeys (Deadwyler S.A. et al., 2007).

Of particular importance are data on the positive effect of orexin A in patients with narcolepsy (Baier P. Ch. Et al., 2008).

The drugs under development that affect the activity of orexin-containing neurons are mainly antagonists of orexin receptors. Some low molecular weight compounds are known, possibly antagonists that specifically bind to OXR1 or OXR2 receptors or simultaneously with both receptors. Some patent applications, for example SmithKline Beecham, describe phenylurea, phenylthiourea and cyanamide derivatives as selective OXR1 antagonists (WO 99/09024, WO 00/47576 and WO 00/47580). SmithKline Beecham recently proposed 2-aminomethylpiperidine derivatives (WO 01/96302), 3-aminomethylmorpholine derivatives (WO 02/44172) and N-aroyl-cyclic amines (WO 02/090355, WO 03/002559 and WO recently in their patent applications). 03/002561) as antagonists of orexin receptors. In WO 01/85693, Banyu Pharmaceuticals disclosed N-acyltetrahydroisoquinoline derivatives in the claims. Other orexin receptor antagonists, such as the novel benzazepine derivatives, are disclosed in WO 02/051838. Actelion Pharmaceuticals Ltd. described in the claims 1,2,3,4-tetrahydroisoquinoline derivatives and their use as non-selective antagonists of the OXR1 and OXR2 receptors (WO 01/68609).

However, antagonists of receptors for orexin A have a different mechanism of action, which causes effects different from those of orexin A.

Active work is underway to create non-protein agonists for the orexin A receptor, but so far it has not been possible to obtain a single approved non-protein drug.

Orexin A is currently synthesized and manufactured by Sigma-Aldrich and Tocris Bioscience for laboratory use only.

However, the use of chemically synthesized molecules, including protein ones, can lead to unpredictable results in the body when used, because as a result of chemical synthesis, some of the molecules can be represented by enantiomers. The concept of enantiomerism plays an important role in pharmaceuticals, since different enantiomers of drugs, as a rule, have different biological activity.

Thus, at present, the urgent task is to develop a method for producing a safe preparation of human orexin A, causing reactions similar to the action of endogenous orexin A. This would allow for controlled, for example, correction of dysfunction of the orexin-containing system, eating behavior, and maintenance of wakefulness.

The authors of the present invention proposed a solution to this problem - a group of inventions. Human orexin A is obtained using the producer strain created by the authors (claim 2 of the formula) obtained on the basis of Escherichia coli BL21 cells (DE3) containing the plasmid DNA created by the authors (claim 1 of the formula) represented by the vector pET151 / D-TOPO containing insertion of a gene encoding a human orexin A protein, characterized by the amino acid sequence of SEQ ID NO: 1, codonally optimized for expression in Escherichia coli cells, the protein being synthesized with a native N-terminus. The authors also developed a method for producing highly purified native recombinant human orexin A (claim 3 of the formula), which consists in culturing the cells of the obtained producer strain using the target protein expression inducer, lysing the cells, washing the inclusion bodies with 0.2 M sodium deoxycholate, and dissolving the bodies inclusions in an 8 M urea solution, refolding orexin A in a refolding buffer (0.1 M Tris pH 8.0, 0.2 mM EDTA) with 0.5 M L-arginine, chromatographic purification of the obtained protein solution on S-Sepharose to Lonkila and S-100 column. The invention claimed to produce recombinant human orexin A, ensure its safety and simplicity in production and use.

Using a preparation of such a protein will allow, for example, a person to maintain a state of wakefulness, which is especially important when performing special tasks and in extreme conditions, when using a safe drug. The pharmacological potential of such a drug is very large, because in everyday conditions, and in a situation of increased stress, the use of such a drug is extremely necessary and relevant.

The invention is illustrated by the following graphic materials:

FIG. 1. Chromatogram of human orexin A on a P ₂₀ RPC HR 16/10 column.

FIG. 2. The average percentage of sleep periods in rats during the daytime (from 11-00 to 14-00) after intranasal administration of saline (gray bars) and orexin A (at a dose of 10 μg / kg of animal weight) (black bars). ** - P <0.05 in comparison with indicators of the duration of sleep in animals of the control group.

FIG. 3. The average percentage of waking periods in rats during the daytime (from 11-00 to 14-00) after intranasal administration of saline (gray bars) and orexin A (at a dose of 10 μg / kg animal weight) (black bars). * -P <0.05 in comparison with indicators of the duration of wakeful periods in animals of the control group.

Table 1. The results of high performance liquid chromatography (HPLC)

Table 2. The degree of activity of rats in the daytime (from 11-00 to 14-00) after the introduction of orexin A and saline intranasally.

The implementation of the present invention.

Example 1. The creation of plasmid DNA encoding human orexin A

The amino acid sequence of human orexin A, SEQ ID NO: 1, was converted to a nucleotide sequence with simultaneous codon optimization for expression in Escherichia coli cells using a specialized program available at http://www.encorbio.com/protocols/Codon.htm.

The synthesis of a gene encoding recombinant orexin A was carried out by lengthening mutually overlapping oligonucleotides according to the described methods (Majumder, 1992).

The selection of optimal primers was carried out by shifting the primer with respect to the template or changing the length of the primer by 3-6 nucleotides. The calculated nucleotide sequences of the primers were chemically synthesized using an ASM-800 DNA synthesizer (BIOSSET, Russia). Next, gene fragments were synthesized using polymerase chain reaction (PCR) and the resulting primers. The synthesized fragments were isolated by gel electrophoresis and cloned into the pGEM-T Easy plasmid vector according to the instructions for the vector. Cloning was carried out using restriction sites Kpnl, SacII, EcoRV, BamHI or through blunt ends. After sequencing, the fragments were excised from the vector using appropriate restriction enzymes or amplified. After the final synthesis step, the obtained orexA gene was cloned into the pGEM-T Easy vector at the restriction sites KpnI and SacI according to the instructions for the vector. Using Sanger sequencing, it was verified that the target gene was obtained.

The resulting gene was cloned in the plasmid pET151 / D-TOPO (Invitrogen ™) according to the instructions for the vector to obtain the protein without add-on at the N-terminus.

Example 2. Creating a producer strain of human orexin A based on Escherichia coli BL21 (DE3) cells

To create a producer strain, we used E. coli cells of strain BL21 with the genotype F-ompT hsdSB (rB-mB-) gal dcm rne131 (DE3), which has lon- and ompT mutations by protease genes, which allows to obtain non-proteolyzed recombinant proteins in large quantities .

Prepared E. coli cells of strain BL21 (DE3) for transformation of the obtained plasmid DNA — competent cells were prepared as follows. Cells were incubated at + 37 ° C overnight in 5 ml of L-broth containing 1% tryptone, 1% yeast extract and 1% sodium chloride. The culture was bred with fresh L-broth 50-100 times and grown on a shaker at + 37 ° C to an optical density of 0.2-0.3 at a wavelength of 590 nm. Upon reaching an optical density of more than 0.3, the culture was diluted with fresh L-broth to an optical density of 0.1 and grown for 30 minutes. Transferred 100 ml of culture to a sterile centrifuge tube and pelleted cells at + 4 ° C at 5000 g for 10 min. The supernatant was discarded, the cells were resuspended in 50 ml of 0.1 M CaCl2, cooled on ice. The cells were incubated for 20 min on ice. Cells were pelleted for 10 min at 5000 rpm, + 4 ° C. The supernatant was decanted, the cells were resuspended in 3 ml of 0.1 M CaCl ₂ , cooled on ice.

3 ml of competent cells were transferred to a cold tube and 5 μg of plasmid DNA was added. Incubated on ice for 1 h. Transfer the tube to + 42 ° C and hold for 5 minutes. The cells were transferred to a flask with L-broth heated to + 37 ° C without antibiotic and incubated at + 37 ° C for 1-1.5 hours. Then the cells were plated on Petri dishes with antibiotic, ampicillin, to select clones containing plasmid DNA . The grown clones were checked for the presence of targeted plasmid DNA using restriction analysis and PCR, and gene expression analysis was performed with 0.2% lactose and IPTG. As a result, a clone of Escherichia coli cells was selected, which was further used to obtain human orexin A (producer strain). The strain was placed in a cell bank.

Expression induction was obtained using IPTG and 0.2% lactose. However, in the second case, a higher yield of targeted protein was achieved at a lower cost, and therefore an inducer of 0.2% lactose was used for further work.

Example 3. Obtaining highly purified native recombinant human orexin A

The cells of the recombinant human orexin A producer strain were scattered with a microbiological loop from a cryoflacon onto the surface of a Petri dish containing LB agar supplemented with 100 μg / ml ampicillin. After incubation in an incubator for 18 h at 37 ° C, the grown colonies were used as seed.

Wednesday PYP-5052 (1% peptone (Gibco, USA), 0.5% yeast extract (Gibco, USA), 50 mM Na ₂ HPO ₄ , 50 mM K ₂ HPO ₄ , 25 mM (NH ₄ ) ₂ SO ₄ , 2 mM MgSO ₄ , 0.5% glycerol, 0.05% glucose and 0.2% lactose) containing ampicillin at a concentration of 100 μg / ml inoculated a single colony of the producer strain. After it was fermented at + 37 ° C in a thermostatically controlled rotary type shaker at 250 rpm for 20 hours until there was no significant change in optical density at a wavelength of 600 nm for 1 h. An aliquot of cells was then selected for analysis by electrophoresis, and the remaining biomass was precipitated by centrifugation at 9000 g.

The precipitated cells were lysed using 3 cycles of sonication for 30 s with a break of 2 minutes on ice. Then, the inclusion bodies were washed three times with 0.2 M sodium deoxycholate, which made it possible to obtain the preparation without admixtures of bacterial endotoxins.

The inclusion bodies were dissolved in an 8 M urea solution, then orexin A protein was refolded in a refolding buffer (0.1 M Tris pH 8.0, 0.2 mM EDTA) with 0.5 M L-arginine.

Chromatographic purification of the resulting protein solution was carried out. About 1200 ml of desalted supernatant was placed on a 20 ml S-Sepharose column, equilibrated with a 20 mM Tris-HCl pH 8.0. The column was washed with 200 ml of 50 mM Tris pH 8.0, the protein was eluted with 160 ml of a linear gradient of 50 mM Tris pH 8.0⋅1M⋅NaCl. The fraction of human orexin A purified on S-Sepharose was placed on an S-100 column (2.5–80 cm), previously equilibrated with phosphate buffer. 1 ml fractions were collected, electrophoretically analyzed in 20% PAG-DDS-Na, fractions with the target protein were combined, and the protein concentration in them was determined by the Bradford method. The column was calibrated using 10 mg bovine serum albumin, ovalbumin and a soybean trypsin inhibitor.

Example 4. Analysis of the obtained orexin A

The uniformity of the orexin A strain, obtained using the method described above, and the plasmid DNA was verified using the HPLC method (High Performance Liquid Chromatography).

The chromatographic homogeneity of the obtained orexin A preparation was checked by reverse phase chromatography (RP) on a P20 column RPC HR 16/10 (Pharmacy, Sweden) in the HPLC mode. The column load was 1.0 mg. Elution was carried out with a concentration gradient of acetonitrile in trifluoroacetic acid (TFA) at room temperature (solution A = 0.1% TFA, solution B - acetonitrile in 0.1% TFA). Protein registration in the eluate was carried out by absorption at 280 nm.

The result of chromatography of a recombinant protein on a P20RPC HR 16/10 column is shown in FIG. 1 and in table 1. Chromatography showed that the obtained preparation of human orexin A is homogeneous, since the protein leaves one peak during OFH, the protein concentration reaches 95.02%.

Example 5. The study of the effectiveness of the obtained orexin A

An analysis was made of the degree of activity of animals within 3 hours after administration of the obtained recombinant orexin A at a dose of 10 μg / kg of animal weight, which allowed us to establish that the obtained compound changes the sleep-wake cycle (Fig. 2, 3). The introduction of the recombinant peptide orexin A leads to an increase in activity, a decrease in the duration of the sleep period (Fig. 2) and an increase in the waking time of rats (Fig. 3) in the daytime (table 2).

The data obtained confirm the effectiveness of orexin A obtained using the method developed by the authors, the producer strain and plasmid DNA. This indirectly confirms the presence of specific activity of orexin A in the protein obtained using the above inventions.

Tab. one.

The method of obtaining recombinant human orexin A, plasmid DNA, producer strain

The method of obtaining recombinant human orexin A, plasmid DNA, producer strain

Sequence listing

<110> Orexal LLC

<120> Method for producing recombinant human orexin A, plasmid DNA, producer strain

<150> RU 2016116370

<151> 2016-04-26

<160> 1

<210> SEQ ID NO: 1

<211> 33

<212> PRT

<213> Homo sapiens

<220>

<223> orexin A

<400> 1

Claims (3)

1. Plasmid DNA for the synthesis of human orexin A with a native N-terminus in Escherichia coli cells, represented by the vector pET151 / D-TOPO, containing the insert of the gene encoding the human orexin A protein, characterized by the amino acid sequence of SEQ ID NO: 1, codonally optimized for expression in cells of Escherichia coli. 2. The strain Escherichia coli BL21 (DE3) pET151 / D-TOPO is a producer of human orexin A, characterized by the amino acid sequence of SEQ ID NO: 1, obtained on the basis of Escherichia coli BL21 (DE3) cells transformed with plasmid DNA according to claim 1. 3. A method of obtaining a highly purified native recombinant human orexin A, characterized by the amino acid sequence of SEQ ID NO: 1, which consists in culturing the cells of the producer strain according to claim 2 using lysed target protein expression inducer, lyse cells, washing inclusion bodies with 0.2 M sodium deoxycholate dissolving inclusion bodies in an 8 M urea solution, refolding orexin A in 0.1M Tris buffer pH 8.0, 0.2 mM EDTA with 0.5 M L-arginine, chromatographic purification of the resulting protein solution is carried out on S-Sepharose column and S-100 column.

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