The method of chromatographic separation and purification of proteins, peptides and their complexes

 

(57) Abstract:

Usage: while gas chromatography insulin. Entity: human insulin obtained from preproinsulin, in which instead of natural methionine between the leader sequence and the proinsulin is arginine, is isolated and purified using ion exchange chromatography. The method allows for the release of insulin without the use of toxic reagents for the simple technology with high yield and high degree of purification. 3 C.p. f-crystals.

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

Currently, one of the important tasks is the problem of creating an effective and safe method of producing human insulin. The strategy of solving this problem is largely determined by the amino acid sequence of preproinsulin. The simplest way is without changing anything, make use of natural primary structure preproinsulin. The disadvantage of this approach is the inevitable use of toxic compounds (bromine cyan) to obtain proinsulin. To easily "cut", for example, trypsin. This approach eliminates brescianello hydrolysis and reducing the processing chain to the final product. Known, for example, a method of hydrolysis of the amino acid sequence of the precursor of human insulin, including processing Clostridium from Clostridium histolyticum in the presence of calcium ions and sulfhydryl reagent at pH 6-9 and processing Arg - derived insulin carboxypeptidase B (EN, 2062301, 1996).

The closest in technical essence and the achieved result is a chromatographic method for isolation and purification of human insulin, including the disintegration of cells containing the hybrid protein, sulfites, separation of the solid phase by centrifugation, dissolved in a buffer solution at pH 7.5, dialyzed on the membrane, renaturation in the presence of 2-mercaptoethanol, the concentration of protein purification using affinity chromatography, lyophilization, dissolution using handinhand, purification on Sephadex G-75, enzymatic cleavage with trypsin and carboxypeptidase B, affinity chromatography of the hydrolyzate and lyophilization (P. Jonasson, Eur.J.Biochem. 236, 656-661, 1996). The authors of this paper modified the procedure for isolation and purification of the final protological activity.

The disadvantage of this method is its complexity and high cost.

The present invention is to develop a simple cost-effective way of allocating human insulin from preproinsulin, in which instead of natural methionine between the leader sequence and the proinsulin is arginine.

The problem is solved by the described method of chromatographic separation and purification of insulin, including the disintegration of cells containing the hybrid protein, sulfites, separation of the solid phase by sublimation, dissolving in a buffer solution with a pH of 7.5, purification by anion-exchange chromatography, preferably on a column of DEAE-Toyperl, balanced trisbuffered pH 7.5 by elution chloride, desalination, renaturation, the protein concentration by ultrafiltration, re anion-exchange chromatography, concentration, enzymatic cleavage with trypsin and carboxypeptidase B, cation-exchange chromatography of the hydrolysate, preferably on a column of S-separate, balanced buffer with a pH of 4.0, when the elution chloride, ultrafiltration and gel filtration.

Replacing expensive stage affinity chromatography, with omowale tech and simple. When hybrid (recombinant) protein is subjected to sulfites formed protein-S-sulfonate. This step allows you to solubilisate and stabilize the protein, effectively carry out its isolation and purification using anion exchange chromatography due to the emergence of 6 negatively charged groups S-SO3.

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

Purified by ultrafiltration and anion exchange chromatography denaturirovannyj recombinant protein, as well as in the prototype, are trypsinolysis. Since the main products of trypsinolysis are di-Arg-insulin and Arg-insulin, further making them insulin, make use of a carboxypeptidase B. Thus, denaturirovannyj recombinant protein can directly turn into insulin, bypassing the stage of receipt of proinsulin. It allows to exclude in the flowsheet stage of cleavage of the recombinant protein with bromine cyan, which is necessary to obtain proinsulin from native preproinsulin. Exclusion from the process flowsheet stage treatment of a number of stages, but also significantly reduces the hazards to the environment during the production of this production. Additionally, the replacement of the chemical cleavage of the recombinant protein to enzyme is very advantageous, as it provides a higher yield and quality of the final product.

After enzyme treatment denaturirovannogo recombinant protein with trypsin and carboxypeptidase B, separation and purification of insulin proposed using cation exchange chromatography.

Fractions with high concentrations of insulin are combined and concentrated by ultrafiltration installation.

Final purification of insulin was performed using gel filtration. Fractions with insulin was liofilizovane.

The above sequence of stages allows to allocate effectively clean the insulin.

The biological activity of the obtained insulin was 29 IU/mg Molecular weight of the obtained insulin controlled using mass spectrometry. N-terminal sequence (9 steps) obtained substance was determined using the method Sanger. Analysis of amino acid sequences showed the presence of two polypeptide chains, prinadlejal producer carry out the transformation of E. coli strain XL1-BLue, using plasmid pInsR. Selected cells carrying plasmid InsR are the producer of preproinsulin.

After preparation of the fermenter to work spend it filling 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 sulfate as - 0.5 g/l, glucose 10 g/l In the environment of the cultivation of the producer strain is maintained pH in the range of 6.7 units, the supply of water and ammonia solution with mass fraction of 12.5%. Dissolved oxygen as consumption support at the level of 35% by changing the mixing speed. When foaming serves sterile aqueous emulsion of antifoam AC-60.

The process is carried out for 3-4 hours to reach the total optical density of at least 10. The amount of biomass is estimated as 20 g/l of cultural liquid. 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 4oC and disintegrate.

In the next step, the resulting suspension was dissolved in buffer solution (OC. The reaction mixture is stirred for 12 hours at a temperature of 20oC.

After sulfatase solution is fed to the sublimation poured into trays and freezing to -70oC. the Drying is carried out in the vacuum (residual pressure in the chamber 0,202 mbar).

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

In the next phase protein absoluut on a column of Sephadex G-25, equilibrated with 50 mm solution of glycine pH of 10.5.

Then hold renaturation protein-S-sulfonate with 2-mercaptoethanol with constant stirring in the cold (4oC). The degree of renaturation of recombinant protein according to high performance liquid ow installing and re-subjected to anion-exchange chromatography. The solution is applied on a column of DEAE-Toypearl, balanced 30 mm Tris-buffer pH 7.5. After application of the sample the column was washed with the same buffer. Recombinant protein elute from the column using a linear gradient of sodium chloride (0 to 0.5 M). Control over the degree of purification is carried out using reversed-phase high-performance liquid chromatography on a column of m-Bondapak C18. Output denaturirovannogo recombinant protein is 25%, the purity of the preparation is 80%.

After that protein concentrate on the installation of filtration and subjected to enzymatic cleavage using carboxypeptidase B and trypsin. The process is carried out at 37oC and after incubation, the mixture is acidified to pH of 4.0 by adding glacial acetic acid. The degree of cleavage of the recombinant protein is 92%.

Then the hydrolysate is subjected to cation exchange chromatography. The solution enzymatic hydrolysate denaturirovannogo recombinant protein applied to the column with S-separate, balanced buffer solution pH of 4.0, containing 50 mm sodium acetate and 6 M urea. After application of the sample the column was washed with the same buffer. The elution of insulin spend a linear gradient of sodium chloride is based chromatography. Fractions with high concentrations of insulin were combined and concentrated. The output stage of about 44%, the degree of purification of the protein not less than 94%.

Next insulin concentrate on ultrafiltration installation. The output stage of 95%.

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

The example confirms the decision of the task is to allocate the human insulin using a simple and reliable ion-exchange technology.

1. The method of chromatographic separation and purification of human insulin, including the disintegration of cells containing the hybrid protein, sulfites, separation of the solid phase, dissolved in buffer solution pH 7.5, renaturation protein, its concentration, enzymatic cleavage with trypsin and carboxypeptidase B, chromatography of the hydrolyzate, wherein after sulfatase solid phase produce sublimation, further purification is carried out by anion exchange chromatography followed by desalting, after renaturation of the protein concentration are ultrafiltrate conducting cation-exchange chromatography of the hydrolysate, ultrafiltration and gel filtration.

2. The method according to p. 1, wherein the anion exchange chromatography is performed on a column of DEAE-Toyperl, balanced Tris-buffer pH 7.5, elution is sodium chloride.

3. The method according to p. 1, wherein the cation exchange chromatography is performed on a column of S-separate, balanced buffer with a pH of 4.0, elution is sodium chloride.

4. The method according to p. 1, characterized in that the gel-filtration is performed on a column of Sephadex G-50SF.

 

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