RU2122549C1 - Chromatography method of isolation and purification of proteins, peptides and their complexes - Google Patents

Abstract

FIELD: biochemistry, medicine, endocrinology. SUBSTANCE: human insulin obtained from preproinsulin where natural methionine located between leader sequence is substituted with arginine is isolated and purified by ion-exchange chromatography. Method ensures to obtain insulin without toxic reagents using. EFFECT: simplified technology, high yield and purity of product. 4 cl, 1 ex

Description

 The invention relates to the field of chromatographic separation of biopolymers and can be used, in particular, for chromatographic isolation and purification of human insulin.

 Currently, one of the important tasks is the problem of creating an effective and safe way to obtain human insulin. The strategy for solving this problem is largely determined by the amino acid sequence of preproinsulin. The easiest 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 produce proinsulin. Another way is to create an artificial linker between the leader sequence and proinsulin, which can be easily "cut", for example, with trypsin. This approach allows you to abandon the bromine cyanide hydrolysis and reduce the production chain of the final product. A known method, for example, is the hydrolysis of the amino acid sequence of a human insulin precursor, comprising treating clostripain from Clostridium histolyticum in the presence of calcium ions and a sulfhydryl reagent at pH 6-9 and treating the Arg derivative of insulin with carboxypeptidase B (RU, 2062301, 1996).

 The closest in technical essence and the achieved result is a chromatographic method for the isolation and purification of human insulin, including the disintegration of cells containing a hybrid protein, sulfitolysis, separation of the solid phase by centrifugation, dissolution in a buffer solution at pH 7.5, dialysis on the membrane, renaturation in the presence of 2 -mercaptoethanol, protein concentration, purification by affinity chromatography, lyophilization, dissolution using guanidine chloride, purification on Sephadex G-75, enzymatic cleavage of t ipsinom and carboxypeptidase B, affinity chromatography and lyophilization hydrolyzate (P.Jonasson, Eur.J.Biochem. 236, 656-661, 1996). The authors of this work modified 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 disadvantage of this method is its complexity and high cost.

 An object of the present invention is to provide a simple, economical method for isolating human insulin from preproinsulin, in which arginine is located between the leader sequence and proinsulin instead of natural methionine.

 The problem is solved by the described method of chromatographic isolation and purification of insulin, including disintegration of cells containing a hybrid protein, sulfitolysis, isolation of the solid phase by sublimation, dissolution in buffer solution with pH 7.5, purification by anion exchange chromatography, preferably on a column with DEAE-Toyperl balanced by Trisbuffer pH 7.5 by elution with sodium chloride, desalination, renaturation, protein concentration by ultrafiltration, repeated anion exchange chromatography, concentration, enzyme active digestion with trypsin and carboxypeptidase B, cation exchange chromatography of the hydrolyzate, preferably on a column of S-Sepharose, equilibrated with pH 4.0 buffer, eluting with sodium chloride, ultrafiltration and gel filtration.

Replacing the expensive stage of affinity chromatography, with which the separation is carried out in the prototype for anion exchange and cation exchange, allows you to make the process more technological and simple. When a fusion (recombinant) protein is subjected to sulfitolysis, a β-S-sulfonate protein is formed. This stage allows you to solubilize and stabilize the protein, effectively carry out its isolation and purification using anion exchange chromatography due to the appearance of 6 negatively charged S-SO ₃ groups.

 Recombinant protein-S-sulfonate is subjected to desalination on a column with Sephadex D-25. Renaturation, as in the prototype, is carried out in a dilute solution using 2-mercaptoethanol.

 Purified by ultrafiltration and anion exchange chromatography, the renatured recombinant protein, as in the prototype, 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 of the stage associated with cyanogen bromide from the technological scheme not only reduces the cost of the final product due to the direct reduction in the number of stages, but also significantly reduces the environmental hazard during the production process. 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 has been proposed 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 above sequence of steps can efficiently isolate pure insulin.

 The biological activity of the resulting insulin was 29 U / mg. The molecular weight of the resulting insulin was monitored 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.

 The invention is illustrated by the following example.

 To obtain a producer strain, the E. coli XL1-BLue strain is transformed using the plasmid pInsR. Selected cells carrying the InsR plasmid are a producer of preproinsulin.

 After preparing the fermenter for work, it is filled with a sterile nutrient medium of the following composition: casein-peptone-20 g / l, yeast autolysate - 14 g / l, potassium phosphate disubstituted anhydrous - 6 g / l, sodium chloride - 5 g / l, magnesium heptahydrate sulfate - 0.5 g / l, glucose - 10 g / l. In the culture medium of the producer strain, the pH is maintained within 6.7 units by feeding an aqueous solution of ammonia with a mass fraction of 12.5%. Dissolved oxygen, as consumed, is maintained at 35% by changing the stirring speed. When foaming serves a sterile aqueous emulsion of antifoam AC-60.

 The process is carried out for 3-4 hours, while the optical density reaches at least 10. The amount of biomass is estimated as 20 g / l of culture fluid. The content of the hybrid protein is 30-35% of the total protein of the cell.

The collection of cells containing the hybrid protein is carried out by centrifugation at 4000 rpm, then the cell suspension is cooled to 4 ^o C and disintegrated.

In the next step, the resulting suspension is dissolved in a buffer solution (pH 9.0) containing 0.02 M ammonium carbonate and 7.5 M urea. Then tetrathionate and sodium sulfite are added to the solution. The reaction mixture is stirred for 12 hours at a temperature of 20 ^o C.

After sulfitolysis, the solution is sent to sublimation, pouring into trays and freezing to -70 ^o C. Drying is carried out in vacuum (residual pressure in the chamber of 0.202 mbar).

 The resulting dry powder was dissolved in 30 mM Tris-buffer pH 7.5 and the protein solution was applied by a peristaltic pump to a DEAE-Toyperl column, previously equilibrated with the same buffer. After applying the sample, the column is washed with the same buffer. In the next step, the recombinant protein is eluted with sodium chloride (linear gradient from 0 to 1M, elution rate 0.06 L / h). Fractions are collected under the control of a flow detector at a wavelength of 280 nm. The composition of the fractions is analyzed by native polyacrylamide gel electrophoresis and ion-exchange high performance liquid chromatography on a column with DEAE TSK5PW.

 In the next step, the protein is desalted on a Sephadex G-25 column balanced with a 50 mM glycine solution, pH 10.5.

Then, protein-S-sulfonate is renatured with 2-mercaptoethanol with constant stirring in the cold (4 ^° C). The degree of renaturation of the recombinant protein according to high performance liquid chromatography is about 51%.

In the next step, the protein is concentrated in an ultrafiltration unit and re-subjected to anion exchange chromatography. The solution was applied to a DEAE-Toypearl column equilibrated with 30 mM Tris buffer pH 7.5. After applying the sample, the column is washed with the same buffer. Recombinant protein was eluted from the column using a linear gradient of sodium chloride (0 to 0.5 M). The degree of purification is monitored by reverse phase high performance liquid chromatography on an m-Bondapak C ₁₈ column. The yield of renatured recombinant protein is 25%, the purity of the drug is 80%.

After this, the protein is concentrated in a filtration unit and subjected to enzymatic digestion using carboxypeptidase B and trypsin. The process is carried out at 37 ^o C and at the end of the incubation, the mixture is acidified to pH 4.0 by adding glacial acetic acid. The degree of cleavage of the recombinant protein is 92%.

 Next, the hydrolyzate is subjected to cation exchange chromatography. A solution of the enzymatic hydrolyzate of the renatured recombinant protein is applied to a S-Sepharose column, equilibrated with a pH 4.0 buffer solution containing 50 mM sodium acetate and 6 M urea. After applying the sample, the column is washed with the same buffer. Elution of insulin is carried out by a linear gradient of sodium chloride (from 0 to 0.5 M). The insulin content was monitored by reverse phase high performance liquid chromatography. High insulin fractions were combined and concentrated. The output at the stage is about 44%, the degree of protein purification is not less than 94%.

 Next, insulin is concentrated in an ultrafiltration unit. The output of the stage is 95%.

 The next step is the gel-filtration of an insulin solution on a Sephadex G-50 SE column. Fractions containing the final preparation are combined and lyophilized. The output of the stage is about 91%, the degree of purification of insulin is more than 96%.

 The presented example confirms the solution of the problem - to isolate human insulin using a simple and reliable ion-exchange technology.

Claims (4)

 1. The method of chromatographic isolation and purification of human insulin, including the disintegration of cells containing a hybrid protein, sulfitolysis, isolation of the solid phase, dissolution in pH 7.5 buffer solution, protein renaturation, its concentration, enzymatic digestion with trypsin and carboxypeptidase B, hydrolyzate chromatography, different the fact that after sulfitolysis the solid phase is isolated by sublimation, further purification is carried out by anion exchange chromatography, followed by desalination, after concentration renaturation e protein is ultrafiltered and subjected to repeated ion exchange chromatography and concentration, and after enzymatic digestion, cation exchange chromatography of the hydrolyzate, ultrafiltration and gel filtration are carried out.  2. The method according to p. 1, characterized in that the anion exchange chromatography is carried out on a column with DEAE-Toyperl, balanced with Tris buffer with a pH of 7.5, the elution is carried out with sodium chloride.  3. The method according to claim 1, characterized in that the cation exchange chromatography is carried out on a column with S-Sepharose, equilibrated with a buffer with a pH of 4.0, the elution is carried out with sodium chloride.  4. The method according to p. 1, characterized in that the gel filtration is carried out on a column with Sephadex G-50SF.