Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/62—Insulins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products

Definitions

• the subject of the invention is a method for producing insulin and derivatives thereof, as well as a hybrid peptide used in this method.
• Diabetes diabetes (diabetes meliitus), a metabolic disease (a group of metabolic diseases) of multifactorial etiology, is one of the most dangerous diseases of society and civilization, also sometimes called the epidemic of the XXI century. Due to serious complications, diabetes can lead to death of the patient if left untreated. In the case when chronic hyperglycemia is caused by insufficient secretion of insulin by the body - type 1 diabetes - the therapy requires continuous use of insulin and/or its analogues. The number of diabetic patients is 5-6% of the population, and the annual increase in the number of diabetics is approx. 4-6% depending on the region of the world.
• biosynthetic human insulin and analogues thereof produced by the transformation of a single chain fusion protein, are the main source of this hormone as an exogenous drug. It is produced in recombinant bacterial systems, particularly in the cells of Escherichia coli (Polish patent No. PL 127 843), or yeast (L Thim et ai., Proc. Nat. Acad. Sci. USA, 83: 6766-6770, 1986).
• the method of producing these drugs is generally described in numerous patent specifications and scientific publications and is based on the overexpression of a gene encoding preproinsulin, i.e. a hybrid polypeptide consisting of a leader protein and proinsulin, i.e.
• the insulin B-chain (or derivative thereof), a linker peptide and the insulin A-chain (or derivative thereof).
• the next manufacturing step is removal of the leader protein and the linker peptide, followed by isolation of the pure hormone.
• One example of such a process is a method of producing human insulin as described in Polish patent No. PL180818, and these methods are being continuously improved (e.g.
• Polish patents PL167810, PL168114, PL177002, PL178466, PL180968, PL183284, PL191901, PL196626, PL198190, PL203195, PL203254, PL210437 and Polish patent applications P.309882, P.310007, P.320644, P.356005, P.374949, P.385586 and P.391975).
• the biosynthetic hu man insulin and analogues thereof are the prima ry therapeutics in the treatment of type 1 diabetes and have significant value in the treatment of type 2 with different glucose control issues.
• the treatment with huma n insulin has brought satisfactory results in most cases for over 30 years, a number of analogues were created for improved metabolic control of diabetes d ue to their faster or prolonged action.
• the sales of insulin analogues have exceeded the half of the global insulin market, while patent protection of those drugs is beginning to expire. This situation allows entering the medica l markets by new manufacturers of such insulins as biosimilar drugs, in accordance with the regulations of the Europea n Union and the United States. For these reasons, efficient biosynthesis methods are becoming important, to provide high expression of the proteins of interest.
• Polish patent No. PL180 818 describes an efficient method to produce recombinant human insulin using E. coli bacterial strain lacking the cytR repressor (described in Europea n patent application No. EP.0303972) transformed with a plasmid with deo operon containing the coding region for the hybrid protein (precursor) including a leader peptide, 62 amino acids long derived from the N-terminus of modified human superoxide dismutase (CuZnSOD) preceded by the N-terminal a mino acid Met and ending with the C-terminal Arg residue linking it to the insulin B-chain.
• CuZnSOD modified human superoxide dismutase
• the insu lin B-chain is linked to the A-chain with a short linker peptide composed of the known linkers Lys-Arg (European patent specification No. EP195 691) or Arg (European patent specification No. EP347 781).
• the aim of the invention was to provide an efficient process for producing insu lin and analogues thereof that would exceed by several percent the yield of the process using the peptide C as a linker protein, as well as the hybrid proteins that are used in this method .
• the subject of the invention is a polypeptide having the amino acid sequence of the formula:
• A is a polypeptide of the insulin A-chain or analogue thereof, prefera bly a sequence selected from SEQ. I D No.: 1-4.
• B is a polypeptide of the insulin B-chain or analogue thereof, preferably a sequence selected from SEQ. I D No.: 5-7.
• n 0 or 1
• X is a leader protein polypeptide, preferably a sequence selected from SOD of SEQ. No.: 8 or U BI of SEQ. No. : 9.
• the polypeptide of the invention has an amino acid sequence selected from : SEQ. I D No.: 10-27.
• the polypeptide of the invention is intended for the production of human insulin by expressing the polypeptide in a strain of Escherichia coli lacking deo repressors and/or deo (cytR) gene containing DNA encoding said polypeptide, wherein said DNA is present in a vector under the control of deoP 1 P 2 promoter, while the hybrid polypeptide comprises a insulin B-chain covalentiy bound to the A-chain via Arg-Arg dipeptide.
• a further subject of the invention is a method for prod ucing huma n insulin comprising microbia l expression of a recombinant protein containing insulin characterized in that it comprises:
• hybrid polypeptide comprising proinsulin by expression of the hybrid polypeptide in a cell of an Escherichia coli strain lacking deo repressor and/or deo (cytR) gene containing DNA encoding the hybrid polypeptide, wherein the DNA is present in a vector under the control of promoter, while the hybrid polypeptide com prises the insulin B-chain covalentiy bound to
• the hu man insulin A-chain and the human insulin B-chain according to the invention is intended to mean both a polypeptide having the naturally occurring amino acid sequence and a seq uence of its known analogue.
• the hybrid peptide is a peptide of the invention as defined above.
• the expression is performed in E. coli strain of the genotype F ara ⁇ (pro lac) rpsL thi cytR recA ⁇ devoid of active cytR repressor protein.
• a plasmid pIBA of the sequence SEQ I D NO: 28 is used as the vector.
• step (a) comprises fermentation in the presence of glucose, glycerol or galactose.
• step (b) comprises:
• step (c) comprises incubating the hybrid polypeptide, preferably in the presence of ascorbic acid, in particular at a concentration of about 2 moles per mole of SH groups present in the mixture, at a temperature of 4 to 37°C for about 1 to 30 hours, preferably about 5 hours, at a pH of 8.5 to 12, preferably at a pH of 11.0 to 11.25.
• step (d) further com prises:
• step (e) fu rther com prises purifying the solution by low-pressure liquid chromatography on Sepharose Q prior to trypsin digestion.
• step (e) further comprises purifying the solution by low-pressure chromatography on DEAE-Sepharose prior to carboxypeptidase B digestion.
• step (e) fu rther comprises purifying the resulting insulin by high-performance liquid chromatography, preferably to a purity of at least 98%, followed by crystallization, filtration a nd drying.
• the insu lin purified at the step (e) is a protein selected from the group comprising: human insulin, human insulin Lys B28 Pro 825 (lispro insulin), insulin Gly A22 Arg B31 (GR insulin), human insulin Ser A22 Arg B31 (SR insulin), insulin Ser A22 Lys B3 Arg K1 (SK3 R insulin), proinsuiin B-ArgArg- A:dezAsn A21 Gly A21 .
• the recombinant preproinsulin according to the invention is understood as a connection of proinsuiin and an additional leader polypeptide, for example ubiquitin or superoxide dtsmutase
• the recombinant proinsuiin is understood as a polypeptide chain, wherein the A and B chains of human insulin or an analogue thereof are connected to any (poly)peptide (linker), that allows them to fold by creating two disulphide bonds between the chains A and B and the third one - within the chain A.
• recombina nt insulin is meant hu man insulin or its recombinant analog of hypoglycaemic activity.
• the particularly efficient method to produce insulin and analogues thereof according to the invention comprising the microbial overexpression of a gene encoding the recombinant hybrid polypeptide, comprising proinsuiin, in cells of Escherichia coli bacterial strains lacking the cytR repressor transformed with an appropriate vector with relevant gene inserted under the control of a deoPlP2 promoter, consists in that the proinsulin produced comprises the insulin B- chain covalently bound to the A-chain via Arg-Arg dipeptide as a linker peptide.
• piBA plasmid has been prepared on the basis of patent specification No.
• PL 180 818 containing a region encoding the deoPlP2 promoter and a gene fragment of h uman superoxide dismutase (SOD, 62 aa) or ubiquitin (UBI) as described in U.S. patents 8,158,382 and 8,956,848, as well as by A. Wojtowicz er a/., M icrobial Cell Fact. 2005, 4: 17 and Microb Cell Fact. 2014; 13 (1): 113. Further, a fragment encoding the relevant proinsulin is in the same reading frame - optionally modified B-chain, the linker peptide and optionally modified A-chain.
• the regulatory elements of the plasmid are derived from pBR322 vector (ATCC 31344).
• the deoPlP2 promoter is derived from the bacterial deo operon composed of four closely related genes that regulate nucleotide and deoxynucleotide catabolism in Escherichia coli bacteria; its sequence was amplified from chromosomal DNA of E. coli K12 strain based on the nucleotide sequence from gene database [GenBank: AP009048]. Based on the strain of E. coli CSH50 ⁇ ATCC: 39111; Cheng et al., Gene, 14 Suppl. 1-2: 121-130, 1981), the E.
• coli I BA strain was derived with the F ara ⁇ (pro lac) rpsL thi cytR recA A " genotype, devoid of active cytR repressor protein by introducing a mutation into the gene using the method described in European patent application No. EP0303972.
• a system was obtained as described in Polish patent PL 180 818 to express the encoded fusion protein in a pIBAINS vector under the control of the deoPlP2 promoter, which is negatively controlled by a chromosomal repressor protein, the product of cytR gene (K. Hammer-Jesperson et al., M olec.
• linker peptide sequences were:
• the thoroughly investigated insulin a nd its derivatives-analogues were: recombinant human insulin (Polish patent specification No. PL128 599) Lys Pro h uman insulin (lispro insulin, Polish patent specification No. PL180 968) Gly A22 Arg 831 human insu lin; (Polish patent specification No. PL219335 or US 08,618,048, GR insulin), Ser A22 Arg B31 human insulin (description of Polish patent application P.219335, SR insulin),
• the £ coli IBA was used as the E. coli host strain to study the yield; the essence of its properties is the inactive gene of cytR repressive protein, which is disclosed a nd described in the literature (e.g. Miller J H., Experiments in molecular genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 1972) or in U.S. patent description 4,480,038 or Polish patent description PL 180 818 for E. coli strains deposited under accession numbers ATCC 69361 and 69363. Other strains widely used in research - E. coli DH5, E. coli DHSa!pha. and E. coli HB101 - were used for comparative purposes.
• the pIBA vector - deoPlP2 promoter, tetracycline resistance, terA Trp tra nscription terminator - was used as the plasmid for protein expression in the yield studies, but for comparative purposes also other vectors were used : pIGALl derivative (Gene Bank AY 424310) - pms promoter, ampicillin resistance, ST1 transcription terminator; pIGALl derivative - pms promoter, tetracycline resistance, terA Trp transcription terminator and pIGALl derivative - deo P1P2 promoter, tetracycline resistance, ST1 transcription terminator.
• pIGALl derivative Gene Bank AY 424310
• PL 180 818 and a modified ubiquitin (UBI), as described in U.S. patent specification 8,158,382 and 8,956,848, were primarily used as the leader protein in the yield studies.
• SOD and UBI represent compounds described respectively in these patent specifications.
• the process for producing each of the insulin which is the subject of the present application is carried out with a classical method of genetic engineering and biotechnology. It consists in that a suitable strain is constructed, which in a process of biosynthesis (fermentation) produces the desired hybrid polypeptide (relevant preproinsulin), which is then transformed into the desired product, purified and isolated.
• the system is universal to produce recombinant human insulin and its studied - slightly different - analogues.
• pu rpose modifications of the recombinant hu man preproinsulin gene were constructed using genetic techniques, such as site-specific mutagenesis reaction .
• Point mutagenesis reaction was carried out using a kit from Stratagene (Cat. No. 200518-5); plasmid DNA of pIBA a nd pIGAL plasmids was used as the template.
• any other DNA comprising the relevant sequence coding for recombined human proinsulin or preproinsulin can be used as the template.
• vectors - were prepared that encoded the studied (pre)proinsulin with a linker (poly) peptide, and elements-DNA fragments encoding various proinsu lins were used for the plasmid construction - consisting of various linker peptides and of various insulin derivatives.
• These vectors were used to transform competent cells of a suitable Escherichia coli strain, for exam ple IBA, DH5a, DH5 or HBlOl, and it is possible to use cells of other E. coli strains or cells of other microorganisms, or other known cell lines suita ble for recombinant protein expression.
• the plasmid containing the specific gene modification of recombinant human preproinsulin was isolated and sequenced to verify the correct transformation a nd nucleotide sequence.
• Single clones of the transformed strains were typically cultured in appropriate medium supplemented with selection antibiotic to prepare the bacterial material for the resea rch ban k, and the samples of bacterial cu ltures and 40% glycerol in the ratio 1:1 were deposited at -70°C.
• Ail research bank strains were subjected to extensive microbiological, biochemical and physicochemical testing, which confirmed their stability and compatibility of their properties with the requirements applicable to industrial banks. The course of subsequent biosynthesis was based on Polish patent specification No. PL 180 818.
• the variants of the recombinant preproinsulin produced in £ coli stra ins were isolated after cell disruption as inclusion bodies, which were separated.
• Hybrid polypeptide with insulin or analogue thereof (corresponding preproinsulin) was isolated from the inclusion bodies, and then subjected to folding (renaturation).
• the resulting solution of folded hybrid protein was subjected to the controlled action of trypsin, similarly as in the case of a number of methods known and described previously (e.g. by Kem mler et al., J. Biol. Chem., Vol. 246, pp.
• the resulting insulin and its analogues were subjected to a process of purification using known methods, mainly low-pressure chromatography, ultrafiltration, and HPLC. From the sufficiently pu rified - meeting pharmacopoeia l requirements - solution of insulin or its analogue, the product was crystallized and dried.
• coli strains was performed in accordance with the general methods of their culture. I noculum culture was ca rried out for approx. 10 hours, until the end of the logarithmic phase of bacterial growth, and production culture for a pprox. 16 hours. The subsequent process of product separation, tra nsformation, and purification was also conducted as described in Polish patent No. PL 180 818 - optimized example 2 a nd 3. I n particular, on completion of the production biosynthesis the resulting broth was cooled to approx. 10°C, £ coli cells were centrifuged, subjected to action of lysozyme a nd Triton X100, and then sonicated with ultrasound or disintegrated with pressure at 5-10°C.
• the inclusion bodies were centrifuged again, washed, and then subjected to renaturation, as genera lly described in exam ple 3A of the Polish patent PL 180 818. Except for the proteins obtained according to Example 6, the further optimized process consisted in subjecting the solution to low-pressure liquid chromatography on DEAE Sepharose following citraconylation and digestion with trypsin or digestion with deubiquitinating protease a nd trypsin. After decitraconylation and precipitation and dissolution of the protein, the solution was subjected to another low-pressure liquid chromatography using-Sepharose Q resin, followed by optional digestion of the linker peptide with carboxypeptidase B.
• weight of the isolated inclusion bodies expressed in g per 1 dm 3 of production culture (wet and/or dry or as dry weight of the slurry in a buffer);
• total protein (after precursor folding) as determined by a spectrophotometric method a nd expressed in AU/dm 3 or H PLC and expressed as % folded precursor in the total protein;
• the invention discloses that the final yield of biosynthetic human insulin and its analogues, using the optimized technological process of biosynthesis, secretion, transformation, a nd purification, depending on the degree of overexpression of the gene encoding preproinsulin in the modified Escherichia coli cells (with inactive cytR repressors that negatively regulate deo operon ⁇ transformed with a specific plasmid (with deoPlP2 promoter) surprisingly depends primarily on the peptide linker used, which links the A and B chains of insulin.
• the basal medium is the basal medium
• ntibiotic tetracycline or ampici!lin
• the flask with the basal medium and antibiotic was inoculated with a single bacterial colony obtained after transformation a nd shaken (220 rpm) at 37°C to an optical density OD 600 of approx. 1.
• the resu lting culture was mixed with the freezing medium (1:1) and frozen at -70°C in 1 ml aliquots.
• the inoculum was prepared in flasks of 250-300 ml in volume filled with 50 ml of inoculum medium. To each flask the following sterile solutions were added:
• antibiotic tetracycline or am picillin, 12.5 mg/ml
• the inoculum medium (components for 1 dm 3 ):
• Step 3 Production culture (NewBrunswIck BioFlo 310 fermenter, 7.5 dm 3 ⁇ starting volume 4 dm 3 )
• the prod uction medium is :
• pH (7.1 +/- 0.1) is controlled by cascade using 16% ammonia.
• the oxygen level in the medium is maintained at a level not lower than 30-35%.
• This parameter is controlled depending on the reading from DO electrode by the increase in impeller speed from 250 to 1000 rpm and in air flow from 5 to 10 dm 3 /min. After reaching the maximum speed of the impeller and the maximum air flow (approx. 7-10 hours) the addition of glucose is stopped; OD 600 value (in the case of E. coli I BA/plBA) is approx. 25-35.
• the biomass is centrifuged using a fixed centrifuge at 6,000-8,000 rpm for 15 minutes and at a tem perature of 4°C.
• the fermented broth may be centrifuged using a flow centrifuge.
• the biomass is resuspended in 1 dm 3 of 50 m M Tris buffer, 0.5 M NaCl and 5 mM beta- mercaptoethanol, pH 7.5, and lysozyme is added to a concentration of 0.43 mg/cm 3 and 0,2 M EDTA to a concentration of 0.5%, then incubated for 30 minutes at room tem perature and subjected to disintegration by sonication or pressure.
• Step 6 - option 1 Sonication.
• the material is cooled to approx. 10°C, divided into 4 parts, Triton is added to 1% and each portion is sonicated for 30 min utes with the amplitude of 33%.
• the sonicated sample is centrifuged at 8,000 rpm for 15 minutes at room temperature.
• the peliet is then resuspended in 1 dm 3 of 50 mM Tris buffer, 0.5 M NaCl and 5 m M beta-merca ptoethanol, pH 7.5 and sonicated again for 10 minutes.
• the inclusion bodies are centrifuged at 8,000 rpm for 15 minutes at room temperature.
• Step 6 - option 2 Pressure disintegration.
• the material is cooled to approx. 10°C, subjected to pressure disintegration (Panda+lOOO, GEA Niro Soavi) at a pressure of 800 bar at a rate that ensures total destruction of the cells (microscopic inspection), and cooling the suspension so that the temperature does not exceed approx. 10°C.
• Step 7 Dissolution of inclusion bodies
• the inclusion bodies were dissolved in adequate amount of 12 mM bicarbonate buffer and 0.2 mM EDTA to obta in absorbance values below 9 at a wavelength of 280 nm.
• the solution is adjusted to a pH va lue of 11.5 ⁇ 0.5 with 2 M NaOH and stirred for 30 minutes at room temperature. Then the pH is adjusted to 10.8 ⁇ 0.4 with 2 M HC1, The solution is clarified by centrifugation at 12,500 rpm for 15 minutes at 4°C.
• the protein After dissolving the inclusion bodies, the protein is renatured by aeration - vigorous stirring for approx. 16-18 h at room temperature. The pH is then adjusted to pH 9.0 with 2 M HCl.
• volume of trypsin A 280 x volume of a solution of [dm 3 ] / 95)
• aprotinin solution with a concentration of 1 mg/mi, added in an amount 21-times less than the volume of trypsin.
• Step 11 Cutting off the leader protein - ubiquitin.
• Step 12 The low-pressure chromatography on a bed of DEAE Sepharose
• the solution at pH 8.6 ⁇ 0.2 is loaded onto a column packed with DEAE resin (200 cm 3 ) a nd eq uilibrated with 0.5 M Tris, pH 8.6 ⁇ 0.2 followed by 20 mM Tris, pH 8.6 ⁇ 0.2.
• the column is washed with buffers at pH 8.6 + 0.2: 20 mM Tris with NaCl until conductivity is 5 + 1 mS, and then 20 mM Tris with NaCl until conductivity is 2 ⁇ 1 mS and with 20 ⁇ 5% isopropanoi.
• the insulin protein is eluted with a buffer at pH 8.6 ⁇ 0.2, consisting of 20 m M Tris with NaCi until conductivity is 5 ⁇ 1 mS and supplemented with 25 + 5% isopropanoi. Step 13. Decitraconylation
• the main fraction e!uted from the colum n is diluted 2 times, cooled to 4°C and acidified with 0.1 M HCI to a pH of 2.9 ⁇ 0.2, then stirred 10 hours.
• Step 14 Precipitation of insulin with zinc chloride.
• the solution of dissolved zinc salt of insulin at pH 8,6 is loaded onto a column packed with the Q resin (SO cm 3 ) and equilibrated with 0.5 M Tris, pH 8,6 and then 20 mM Tris, pH 8.6.
• the column is washed with 20 mM Tris buffer with 20 ⁇ 5% isopropanol, pH 8.6, and then the insulin protein eluted with a buffer at pH 8.6 consisting of 20 mM TRIS with NaCI until conductivity is 3 ⁇ 2 mS and with 25 ⁇ 5% isopropanoi.
• the separation is carried out on a Kromasil C8 or C18 column using an acetonitriie gradient:
• Mobile phase A 2,000 cm 3 of a 0.2 M sodium sulfate solution with 440 cm 3 of acetonitriie, pH 2.3 and a temperature of 25-30°C;
• Mobile phase B 1,250 cm 3 of a 0.2 M sodium sulfate solution with 1,250 cm 3 of acetonitriie, pH 2.3 and a temperatu re of 25-30°C;
• the solution after reaction with carboxypeptidase B or a main fraction after purification on the Q Sepharose resin, following a double dilution and adjustment of the pH to 3, is purified in portions by loading onto the column a single volume of solution having a total protein content of 200 AU, and then the main fraction is collected, so that product purity (HPLC) is at least 98%.
• Step 18 Precipitation of insulin with zinc chloride.
• the combined main fractions from the high-pressure chromatography are diluted 2 times and 1 M ZnCI 2 is added in a proportion of 3 cm 3 of zinc chloride per 150 cm 3 of the sample, and then the pH is adjusted to 6 + 1, followed by stirring for 1 hour.
• the suspension is then centrifuged at 9,000 rpm for 15 minutes at 4°C and the pellet is resuspended in 10-15 cm 3 of water.
• Step 19 Chromatography on a bed Sephadex G-25 and freeze-drying.
• the slurry is adjusted to pH 3, filtered through a 0.22 ⁇ m filter and loaded onto a column filled with Sephadex G 25 resin (120 cm 3 ). Before loading the column is equilibrated with 5 m M ammonium acetate, pH 4, and the protein is eluted with 5 m M ammonium acetate, pH 4. The resulting material is subjected to freeze-drying.
• step I the production of biosynthetic lispro insulin was performed following steps 1-6, 6b, 7-10 and 12-19 a nd for the expression system 1.2. and 1.5. additionally step 11 (Table 1. Lispro insulin).
• the process was studied in 20 variants of 8 protein expression systems. The data obtained shows that the strains of Escherichia coli DH5a, DH5 and H B101, which perform well in laboratory conditions, with piasmids with deoPlP2 and pms promoters are not suitable for production culture in the minimal medium (system 1.3., 1.4., 1.5,, 1.6. and 1.7.).
• strains are characterized by low growth on minimal technological medium, demonstrating a much lower optical density value in the production fermenters.
• a similarly low feasibility for production is shown by the IBA strain system with disrupted cytR repressor and pIGAL piasmid (system 1.8.), characterized by relatively better growth, but low content of inclusion bodies (microscopic observations).
• the best expression yield is shown by the I BA/pl BA strain system, described in Polish patent specification No. PL 180 818, which was tested in 4 linker peptide variants and 2 leader protein variants (system 1.1. and 1.2.). All have good, comparable growth parameters in the minimal medium, but differ in weight of inclusion bodies and relative yield of the fusion peptide renaturation, and - consequently - the final product yield.
• the highest yield of lispro insulin was obtained using the E. coli IBA/pl BA expression system producing A-chain linked to the B-chain of lispro insulin with ArgArg dipeptide (Table 1, Example No. 1.1.1. and 1.2.1. ⁇ , exceeding the performance of other systems by at least 13-15%.
• SK3R insulin biosynthetic SK3R insulin
• the data obtained shows (Table 5. SK3R insulin) that the IBA/plBA strains system is characterized by the best expression yield, showing good, comparable growth parameters in the industrial minimal medium and differing in the weight of inclusion bodies produced and the relative yield of renaturation .
• the highest yield of human insulin was obtained using the E. coli IBA/plBA expression system producing A-chain linked to the B-chain of human insulin with ArgArg dipeptide (Table 5, Example No. 5.1.1. and 5.2.1.).
• the difference in performance between the ArgArg dipeptide variant of the system and LyzArg is 13-22%, and is comparable to the improvement in system performance described in the Polish patent No. PL 180 818 for LizArg dipeptide and Arg in comparison with the C-peptide, which is 23% and 17%, respectively.
• the pIBA/I NS plasmid of 4354 base pairs in size is composed of the following regulatory sequences and genes:
• the structure of the pIBA plasmid is schematically shown in Figure 1, and its nucleotide sequence and amino acid sequence in Figure 2.
• the structure of the pI BA/INS plasmid comprising an exemplary gene encoding recombinant human insulin protein, wherein the A-chain is linked to the B-chain with LysArg dipeptide is schematically shown in Figure 3, and its nucleotide sequence and amino acid sequence in Figure 4.

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