RU2122581C1 - Strain escherichia coli xli - blue/pinsr - a producer of preproinsulin - Google Patents

Abstract

FIELD: biotechnology, medicine, endocrinology. SUBSTANCE: strain-producer of preproinsulin is obtained by transformation of the strain E. coli XLI-Blue using plasmid pInsR and cells carrying this plasmid are removed. Strain produces preproinsulin at high yield and property that ensures to obtain insulin by enzymatic cleavage. Invention can be used for insulin production. EFFECT: strain indicated above, increased yield of product.

Description

 The invention relates to the field of microbiological and pharmaceutical industries and can be used to create drugs.

 The creation of a highly effective strain producing preproinsulin is one of the most important biotechnological problems, the solution of which has been the subject of numerous studies over the past 10-15 years. There are two fundamentally different paths: the first path is the creation of systems secreting preproinsulin; the second way is intracellular accumulation of the final expression product in inclusion bodies. Each of these paths has its advantages and disadvantages. In the first case, the final product enters the extracellular space. This simplifies the process of isolation and purification of preproinsulin. However, the level of expression of the final product in such systems is low. In systems of the second type, expression is much higher, however, the localization of the recombinant protein in inclusion bodies requires a large technological outlay for its isolation.

 Another problem is the choice of technology for insulin production. The way to solve this problem is largely determined by the amino acid sequence of preproinsulin. One way - without changing anything, take advantage of the natural primary structure of preproinsulin. The disadvantage of this approach is the inevitability of using a toxic compound (bromine cyan) to cleave the leader sequence. Another way is to create an artificial linker between the leader sequence and proinsulin, which can be easily "cut" with trypsin. This approach allows you to abandon the bromine cyanide hydrolysis and reduce the production chain of the final product.

 The closest in technological essence and the achieved result to the proposed invention is the creation and use of the above insulin producing E. coli strain (Nilsson et al., Integrated production of human insulin and its c-peptide J. Biotechnology 1996, 48, 41-50) . Distinctive features are: 1. Use of E. coli 017 strain; 2. An additional site responsible for IgG binding, which enabled the authors of this work to modify the procedure for isolation and purification of the final product so that, along with insulin, it was possible to isolate peptide C, which in their opinion has pronounced biological activity.

 The present invention is the creation of a strain producing preproinsulin with high yield and characteristics that allow you to get insulin by simple and effective technology.

 The essence of the invention is to create a strain of E. coli XLI-Blue / pInsR, a producer of preproinsulin, characterized by the presence of recombinant plasmid DNA pInsR.

 The producer strain Escherichia coli XLI - Blue / pInsR is characterized by the following features.

 Morphological signs. The cells are rod-shaped, gram-negative, non-spore.

 Cultural signs. Cells grow well on simple nutrient media. On the Difco agar, the cells form round smooth colonies with even edges. When growing in a liquid nutrient medium, they form an intense smooth turbidity.

Physico-chemical signs. Cells grow in the temperature range from 8 ^o C to 40 ^o C, the optimum pH is from 6.8 to 7.0. Mineral salts, casein, peptone, yeast autolysate, etc. are used as sources of nitrogen, carbon, and other necessary components.

 Antibiotic resistance. Cells are resistant to tetracycline and ampicillin.

 The E. coli XLI-Blue / pInsR producer strain differs from the E. coli XLI-Blue recipient strain by the presence of recombinant plasmid DNA pInsR, which confers additional resistance to ampicillin.

 E. coli XLI-Blue / pInsR cells are a producer of preproinsulin. During the induction of isopropyl-beta-D-thiogalactoside, an intensive synthesis of this protein occurs, which accumulates in the form of inclusion bodies. The yield of preproinsulin is more than 30% of the total protein of the cells.

 The invention is as follows.

 To obtain a producer strain, the E. coli XLI-Blue strain is transformed using the plasmid pInsR (shown at the end of the description) (patent application N 97114220/13 of 08/28/97). Selected cells carrying the InsR plasmid are a producer of preproinsulin.

 At the next stage, the required amount of biomass is grown. To control the growth dynamics of the culture, samples are periodically taken and controlled. A few hours (depending on the volume of the fermenter) before the end of the process of operating the culture make the inductor.

 After completion of the fermentation process, the cell suspension is centrifuged. Selected cells destroy.

Next, the recombinant protein undergoes a sulfitolysis reaction to form a recombinant protein - S-sulfonate. This type allows you to stabilize the protein and effectively carry out its isolation and purification using anion exchange chromatography due to the appearance of 6 negatively charged S-SO ₃ groups.

 Then, the recombinant protein S-sulfonate is subjected to desalination on a Sephadex G-25 column and renaturated in a dilute solution with 2-mercaptoethanol.

 The purified, renatured recombinant protein is subjected to trypsinolysis. Since the main products of trypsinolysis are di-Arg-insulin and Arg-insulin, their further conversion to insulin is carried out using carboxypeptidase B. Thus, the renatured recombinant protein can be directly converted to insulin, bypassing the stage of obtaining proinsulin. This is what allows us to exclude in the technological scheme the step of splitting the recombinant protein with cyanogen bromide, which is necessary for obtaining proinsulin from native preproinsulin. The exclusion from the technological scheme of the stage associated with bromine cyan not only reduces the cost of the final product by directly reducing the number of stages, but also significantly reduces the harmfulness of this production. In addition, replacing the chemical cleavage of the recombinant protein with an enzymatic one is very beneficial, as it provides a higher yield and quality of the final product.

 After enzymatic treatment of the renatured recombinant protein with trypsin and carboxypeptidase B, the isolation and purification of insulin is carried out using cation exchange chromatography.

 High insulin fractions are combined and concentrated in an ultrafiltration unit.

 The final purification of insulin was performed using gel filtration. Fractions with insulin were lyophilized.

 The biological activity of the resulting insulin was 29 U / mg. The molecular weight of the resulting insulin is determined by mass spectrometry. The N-terminal sequence (9 steps) of the obtained substance was determined using the Sanger method. Analysis of amino acid sequences showed the presence of only two polypeptide chains belonging to insulin.

Claims (1)

 Strain Escherichia coli XLI - Blue / pInsR - producer of preproinsulin.

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