WO2009154514A2

WO2009154514A2 - Producer strain of soluble recombinant human hepsin (hpntm -a)

Info

Publication number: WO2009154514A2
Application number: PCT/RU2009/000300
Authority: WO
Inventors: Евгений Сергеевич СЕВЕРИН; Сергей Евгеньевич СЕВЕРИН; Екатерина Михайловна КУЗНЕЦОВА; Мария Владимировн САВВАТЕЕВА; Михаил Петрович КИРПИЧНИКОВ
Original assignee: Aho "Иhctиtуt Молекулярной Диагностики"
Priority date: 2008-06-19
Filing date: 2009-06-15
Publication date: 2009-12-23
Also published as: EA200801823A1; EA011695B1; WO2009154514A3

Abstract

The invention relates to biotechnology and can be used for producing soluble hepsin which can be used in public health for prostate cancer diagnosis and for treating oncological patients, as well as for producing antibodies that are able to bind hepsin in the human blood.

Description

STRAIN PRODUCER OF THE SOLUBLE FORM OF HUMAN RECOMBINANT HEPSIN (HPNTM ^A ).

The field of technology.

The invention relates to the field of biotechnology and can be used in the microbiological industry for the production of recombinant hepsin serine protease for use in healthcare, in particular for the diagnosis and treatment of prostate cancer.

The prior art.

At present, prostate cancer (PCa) is one of the most common tumor diseases in men, the frequency of which is directly related to age: for example, starting from 45 years old, the incidence of prostate cancer increases exponentially and in men aged 70 years and above it is more than 60%.

In this regard, the task is to select from the total mass of potential markers of prostate cancer those that have the highest prognostic value and specificity and can be used in clinical practice without complicating the method of use for diagnosis and its significant cost increase.

It is known from the prior art that hepsin can serve as such a marker (1). Hepsin is a serine protease that is not expressed by healthy prostate cells, but is present in significant amounts on the surface of prostate cancer cells. This circumstance allows the use of hepsin in the diagnosis and treatment of the disease with a high degree of certainty (2). Another important feature of hepsin is associated with its enzymatic activity, which determines its role in the process of oncogenesis of a prostate tumor. It was shown in experimental data that when using monoclonal antibodies to bind hepsin on the cell surface, the tumor's ability to metastasize was significantly reduced (3). Therefore, a test based on the determination of this particular function (enzymatic) most fully characterizes the stage of the disease and the development of the tumor process.

Disclosure of the invention.

To solve the problem of using hepsin for the diagnosis of prostate cancer and the stage of this disease, it is necessary to create a soluble producer strain hepsin, since it provides for the subsequent receipt of protein in active form.

The hepsin producing strain is not known from the prior art, but to date, a full-sized form of inactive recombinant hepsin with glutathione c transferase (Abpova, cat Na H00003249-P01) has been obtained in the world. This product is used as a concentration standard in the formulation of ELISA and Western blot techniques. However, it is not an active form of protein and is not suitable for the implementation of enzymatic reactions specific for high-grade hepsin and, accordingly, is not applicable for the diagnosis of prostate cancer.

The E. CoIi producing strain carrying the pHPNTM ^"A plasmid DNA with an integrated gene encoding human hepsin was obtained by BL21 (DE3) CodonPlusRIL calcium cell transformation method. The producing strain was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) under NsB-100

Storage conditions.

The strain can be stored without loss of useful properties at a temperature of -70 ° C in an environment of the following composition: 1% bacto-tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.0,

15% glycerol, 7% DMSO for at least 6 months with mandatory reseeding at least once every 2 months.

Cultural and morphological characteristics of the strain.

Cells are short (length 1-3 microns, width 0.5-0.8 microns) polymorphic movable gram-negative bacilli that do not form spores.

On meat and peptone broth give abundant growth with a significant turbidity of the environment; the precipitate is small, grayish in color, easily broken. They form a parietal ring, the film on the surface of the broth is usually absent. On meat and peptone agar colonies are transparent with a grayish-blue tint, easily merging with each other. On medium Endo they form flat red colonies of medium size with a dark metallic sheen. Physiological and biochemical properties.

Optional aerob. The temperature range of growth is 18-38 ° C. The optimum growth temperature is 37 ° C. It grows at pH values of 5.0-8.0. The optimum pH of growth is 6.5-7.5. Negative Fogis-Paraskau reaction; positive reaction to the formation of indole; negative reaction to the formation of hydrogen sulfide; negative reaction to citrate; negative reaction to the formation of malonate; negative reaction to the formation of urea; positive reaction to lysine decarboxylase; negative reaction to ornithine; positive reaction to gas formation; negative reaction to lactose synthesis; positive reaction to the breakdown of mannitol.

The species E. CoIi, to which the strain BL21 (DEЗ) CodonPlusRIL (Stratagep, US Patent No. 6,706,525) is assigned, is not listed as pathogenic in the Regulation on the Procedure for Recording, Storage, Handling, Dispensing and Transfer of Bacteria, Viruses, Rickettsia Cultures, fungi, protozoa, mycoplasmas, bacterial toxins, poisons of biological origin *, is not in the lists of pathogenic bacteria according to the Regulation of the Ministry of Health and in the lists of pathogenic bacteria in the Official Journal of European Communities NC 217/3237, 08.24.92.

Characteristic strain:

Family Epterobastereasae;

Genus Esherisia;

View of Escherichia coli; Origin of Protogepe; the source of excretion is the gastrointestinal tract of cattle; genotype: E. coli B F ^"otrT hsdS (r _in ^~ MW ^~) dcm ⁺ TEF dal λ (DEZ) epdA Nte [ardU HeY leiW Cam ^r]; Biotechnological characteristic.

The producer strain contains plasmid DNA derived from the vector pET28a

Figure 1 presents the physical map of the plasmid pHPNTM ^"Ai , nucleotide and amino acid sequences.

General information about the cloned fragment, size and genes included in its composition

Included genes: extracellular part of hepsin (5128 bp - 6246 bp), thrombin cutting site (5464 bp - 5481 bp), 6 Нis-tag (5089 bp - 5106 bp), site for cutting with factor X (5107 bp - 5127 bp) Genetic map of the cloned fragment (size - 1175 bp): Nсol

5051 ATGGGCAGCC GСGGTAGSSA TSATSATSAT

TASSSSGTSGG SGSSATSGGT AGTAGTAGTA 5101 SATSASAGSG STAGSATTGA GGGTSGTSSG STGTASSGAG

TGCAGGTSAG

GTAGTGTSGC GATSGTAAST SSSAGGCAGGС GASATGGGTS ASGTSSAGTS 5151 STSTGСGGAS GSTSGGSTSA TGGTSTTTGA SAAGASGGAA

GGGACGTGGC

GAGASGССТG СGАГССГАГТ АССАГАААСТ GTTSTGSTST SSSTGSASSG 5201 GGSTGSTGTG STSSTSGСGС ТССААГССА GGGТAGССGG

ASTCAGSTGC

SSGASGASAS GAGGAGGGGG AGGTTGСGGT SSSATSGGCC TGAGTSGASG 5251 GAGGAGATGG GSTTSSTSAG GGSASTGASS SASTSSGAGS

TGGASGTGСG

STSTSTSTASS SGAAGGAGTS SSGTGASTGG GTGAGGSTCG ACSTGCACGC 5301 AACGGСGGGC GCCAATGGCA SGTSGGGSTT STTSTGTGTG

GASGAGGGGGA

TTGSSGSSSG SGGTTASSGT GSACSSSSGAA GAAGASASAS STGSTSSSST 5351 GGSTGSSSSA SASSSAGAGG STGSTGGAGG TSATSTSSGT

GTGTGATTGC

SSGASGGGGT GTGGGTSTSS GASGASSTSS AGTAGAGGCA SASASTAASG

5401 СССАГАГGСС GTTTCTTGGC СГССАТСТГС СААГАСТГТG

GCCGCAGGAA

GGGTSTSSGG SAAAGAASSGGСGGTAGASG GTTSTGASAS SGGСGTSSTT 5451 GSTGSSSGTG GASSTGGTGС SGСGСGGСAG SGTGGGAGGGС

CGGGASASSA

СGАСGGGСАС STGGASSASSG GСGСGSSGTS GCASSSTSSG GSSSTGTGGT 5501 GSTTGGGSSG GTGGSSGTGG SAAGTSAGSS TTSGSTATGA

TGGAGCASAS

СGААСССGGС САСCGGСASS ГТТСАГТСGG ААГСGATAST АССТСGТGТG

BamHI 5551 CTCTGTGGGG GATCCCTGCT CTCCGGGGAC TGGGTGCTGA CAGCCGCCCA

GAGACACCCC CTAGGGACGA GAGGCCCCTG ACCCACGACT GTCGGCGGGT

5601 TTGCTTCCCG GAGCGGAACC GGGTCCTGTC CCGATGGCGA GTGTTTGCCG

AACGAAGGGC CTCGCCTTGG CCCAGGACAG GGCTACCGCT CACAAACGGC

Pstl

5651 GTGCCGTGGC CCAGGCCTCTTTACACGGTC TGCAGCTGGG

GGTGCAGGCT

CACGGCACCG GGTCCGGAGA GGGGTGCCAG ACGTCGACCC CCACGTCCGA

5701 GTGGTCTACC ACGGGGGCTA TCTTCCCTTT CGGGACCCCA

ACAGCGAGGA

CACCAGATGG TGCCCCCGATAGAAGGGAAA GCCCTGGGGT TGTCGCTCCT 5751 GAACAGCAAC GATATTGCCC TGGTCCACCT CTCCAGTCCC

CTGCCCCTCA

CTTGTCGTTG CTATAACGGG ACCAGGTGGA GAGGTCAGGG GACGGGGAGT 5801 CAGAATACAT CCAGCCTGTG TGCCTCCCAG CTGCCGGCCA

GGCCCTGGTG

GTCTTATGTA GGTCGGACAC ACGGAGGGTC GACGGCCGGT CCGGGACCAC 5851 GATGGCAAGA TCTGTACCGT GACGGGCTGG GGCAACACGC

AGTACTATGG

CTACCGTTCT AGACATGGCA CTGCCCGACC CCGTTGTGCG TCATGATACC

Aval

5901 CCAASAGGGSS GGGGTASTSS AGGAGGGSTSG AGTSSSSATA ATSAGCAATG

GGTTGTSSGG SSSSATGAGG TSSTSSGAGS TSAGGGGTAT TAGTSGTTAS

5951 ATGTSTGCAA TGGСGSTGAS TTSTATGGAA ASSAGATSAA GCCCAAGATG

TASAGASGTT ASSGCGASTG AAGATASSTT TGGTSTAGTT SGGGTTSTAS

Aval

6001 TSTSTGTGSTG GSTASSSSGA GGGTGGCATT GATGCSTGSS AGGGСGASAG

AAGASASGAS SGATGGGGST SSSASSGTAA STASGGASGG TSSSGSTGTS 6051 CGGTGGTCCC TTTGTGTGTG AGGACAGCAT CTCTCGGACG

CCACGTTGGC

GCCACCAGGG AAACACACAC TCCTGTCGTA GAGAGCCTGC GGTGCAACCG 6101 GGCTGTGTGG CATTGTGAGT TGGGGCACTG GCTGTGCCCT

GGCCCAGAAG

CCGACACACC GTAACACTCA ACCCCGTGAC CGACACGGGA CCGGGTCTTC

6151 CCAGGCGTCT ACACCAAAGT CAGTGACTTC CGGGAGTGGA

TCTTCCAGGC

GGTCCGCAGA TGTGGTTTCA GTCACTGAAG GCCCTCACCT AGAAGGTCCG

6201 CATAAAGACT CACTCCGAAG CCAGCGGCAT GGTGACCCAG CTCTGA

GTATTTCTGA GTGAGGCTTC GGTCGCCGTA CCACTGGGTC GAGACT

Amino acid sequence:

mgsrgshhhhhhsasiegrrlurvqvssadarlmvfdktegtwrllsssrsparvaglsseemgflralthseldvrtagap gtsgffsvdegrlrhtqrllevisvsdsrrgrflaaisqdsgrrklrvdlvrrgsvggrdtslgrwrwqvslrudgahlsggsllsgdwvlt aahsfremr / lsrwrvfagavaqasrhglqlgvqavvuhggulrfrdrpseepspdialvhlssrlrlteuiqrvslraagqalvd gkistvtgwgptquugqqagvlqearvriispdvspgadfugpqikrkmfsagureggidasqgdsggrfvsedsisrtrrwrlsg ivswgtgsalaqkrgvutkvsdfrewifqaikthseasgmvtql

The useful substance produced is the extracellular fragment of hepsin, an Il type transmembrane serine protease.

It has all the characteristic features of serine proteases, hydrolyzes the peptide bond with arginine in the position Ls P ₂ Pz Arg I, where P ₂ is preferably Asp, Leu or Thr, P ₃ preferably Ls or GIn (4).

- productivity level 20%

An example embodiment of the invention.

The pHPNTM ^"A plasmid DNA carrying the gene encoding human hepsin was created in accordance with the map shown in Fig. 1 and the given nucleotide and amino acid sequences using standard molecular biology methods known from the scientific and technical literature (5). The resulting plasmid containing the HPNTM gene ^"A , was transformed into E. coli cells of strain BL21 (DE3) CodonPlus RIL

Analytical expression of the target protein was carried out using a chemical inducer. For this, E. coli colonies carrying the target protein gene, grown in liquid nutrient medium LB with antibiotic in test tubes for 15 + 2 hours at a temperature of 37 ° C with constant shaking in a rocking chair. 0.5 ml was added to 250 ml flasks containing 50 ml of LB antibiotic liquid. They were grown at a temperature of 37 ° C with constant swaying to an optical density of 0.5 + 0.1 (OD540). Then added a chemical inducer - IPTG (isopropyl-β-D-thiogalactopyranoside) and increased further for 3 hours. Biomass was collected by centrifugation in 50 ml plastic tubes (OPH-8 centrifuge, 4.000 rpm for 8 minutes).

Conducted electrophoretic analysis of cell protein in a polyacrylamide gel under denaturing conditions. For this, 40 μl of water was added to an aliquot of 10 mg cells, mixed on a shaker and 40 μl of 2x sample buffer for protein denaturing electrophoresis was added. The sample was heated at 95 ° C for 4 minutes and applied to a denaturing gel with an acrylamide concentration of 12%. After electrophoretic separation, the gel was washed with distilled water, and then stained with a paint containing Coomassie dye. Excess paint was washed with a solution containing ethanol and acetic acid.

The analysis of the presence of the target protein was carried out visually, comparing the set of cellular proteins in uninduced cells and cells induced by IPTG. To determine the molecular weight of the obtained target protein, a set of proteins with known molecular weights was used.

The amount of the target protein was analyzed in relation to the total cellular protein. For this, the gel was scanned on an Ersop 1660 Pho scanner. The resulting image was processed using the Operadsap program.

The expression results show a high level of synthesis of the target protein, the target protein is at least 20% of the total cellular protein (an average of 20-25%).

Information sources:

1. Liteus SP, Loeb KR, Hagen FS, Kurashi K, Davie EW. And he began to try-lik-serip rotease (hersip) with and puttricembrape domap expresed hum humive update apothellas. Biochemistru. 1988; 27 (3): 1067-74.

2. Tanimoto H, Yan Y, Clarke J, Korourian S, Shigemasa K, Parmleu TH, Parcham GP, O'Vriep TJ. Hersip, and the well surfase, the serotype, the idipepti, the hell, the overseas, is the overpower sapper. Sapser Res. 1997; 57 (i4): 2884-7.

3. Huap JA, Skhpeider D, Tou P, Lip R, Neptop A, Zhu Y, Fister S, Vogel D, Miptzer B, Opter H, Light D, Parr R, Rolokoff M, Whitlow M, Wu Q, Parr Pharmacy Peutralizip hersip rotease assistance to imprt sell growth but iphibit ipvsiof of grow apd ovariap tumors sulture. Sapser Research 2006; 66 (7): 3611-9.

4. Sherter, Pirer DE, Aaron W, Gabiele T, Cutler G, Cao P, Batt AS ₁ Cho Y, Craick CS, Walker N, Meininger D, Noe T, Austin RJ. Neutrality of the success is a pre-defined type of vitro substitute for a humper type, and a medium-sized serialized serialized serialized apresive. Biochemistor 2005; 390 (Pt 1): 125-36.

5. Molecular Сlopipg: And Laboru Shpua \ (Third Ediopi) By Joserh Sambrok, Reter Massalim, David Rissell

Claims

CLAIM

The Escherichia coli bacterial strain pHPNTM ^'A is a producer of the soluble form of recombinant human hepsin (HPNTM ^"A ).