[Optimization of the industrial production of the recombinant precursor of human insulin].

Conditions were found at the analytical level for the solubilization of a recombinant insulin precursor from inclusion bodies in different buffer systems at a wide pH range in the presence of different reducing (dithiothreitol, dithioerythritol) and chaotropic agents (urea, guanidine hydrochloride) and the subsequent renaturation with the use of redox pairs (cysteine-cystine, oxidized glutathione-reduced glutathione, and others). The scaling of the method for the production of the active substance of genetically engineered human insulin has been performed.

[Validation of a method for monitoring of genetically engineered human insulin manufacture].

A method for monitoring the manufacture of genetically engineered human insulin by HPLC was developed. The method was validated by the estimation of its linearity, correctness, accuracy, specificity, and stability; the limits of detection and quantitative assessment were also determined. It was proven that HPLC analysis enables reliable and reproducible results to be obtained and can be used for monitoring insulin manufacture.

Size-exclusion chromatography based on silica-diol for the analysis of the proinsulin fusion protein.

Size-exclusion chromatography based on silica-diol sorbent was employed to analyze the recombinant proinsulin fusion protein obtained during the process of refolding and the following ion-exchange purification. The assay was qualified as a control method estimating its accuracy, precision, linearity, limit of detection, limits of quantitation, specificity, and robustness. The results show the reliability for the intended use.

[Analysis of major antigenic determinants in Mistletoe lectin I]. / Analiz osnovnykh antigennykh determinant Mistletoe lectin I.

Antigenic determinants of Mistletoe Lectin I, a toxin from Viscum album were predicted on the basis of the primary amino acid sequence of the protein. Based on the results of analysis, the peptide FPGGSTRTQARS, which corresponds to the 144-155 segment of the viscumin A-chain, was synthesized. The peptide was tested in enzyme-linked immunosorbent assay with monoclonal antibodies against the viscumin A-chain obtained previously. The peptide reacted with antibodies with a low affinity and did not inhibit the binding of viscumin molecule to any of the antibodies. Analysis of the peptide by 1H-NMR spectroscopy in aqueous solution was performed. The three-dimensional structure of the 144-155 segment in the native protein globule was shown.

[A retrospective examination of a hepatitis focus in plasma donors]. / Retrospektivnoe obsledovanie ochaga gepatita u donorov plazmy.

Data are presented on the retrospective epidemiological serological investigation of episodes of hepatitis-like diseases among plasma donors in Krasnoyarsk. A mixed structure of the focus was established with prevalence of B- and non-A-, non-B hepatitides. Markers of recent infection with HB virus were recorded in 43.6% of sick donors, at the same time no donors had IgM antibodies to hepatitis A evidencing the acute stage or early convalescence. The use of the insufficiently sensitive passive hemagglutination test for HBsAg identification has resulted in incomplete detection of donors-carriers of HB virus.

Displacement effect during HPLC preparative purification of human insulin.

HPLC plays a key role in the preparative purification of human insulin. A21-desamidoinsulin is one of the impurities that possesses the chromatographic behavior similar to that of insulin and hence separation from this by-product is rather difficult at the process scale. During the optimization of insulin reversed-phase HPLC purification, when a column was sufficiently overloaded, the effect of displacement of A21-desamidoinsulin molecules from active groups of sorbent by insulin ones was observed. It was suggested that monocarboxylic acid and organic modifier in mobile phase are responsible for the esterification during which the formed ester promotes the displacement effect. This effect was studied in order to optimize the purification of human insulin at the process scale.

[Gene engineered insulin and its pharmaceutical analogs].

Studies of replacement therapy of diabetes mellitus resulted in introduction of series of forms of insulin and new insulin analogs which exhibit better control of blood glucose level. The present paper deals with basic tendencies in this field.

Apoprotein B degradation is promoted by the molecular chaperones hsp90 and hsp70.

Apoprotein B (apoB) is the major protein of liver-derived atherogenic lipoproteins. The net production of apoB can be regulated by presecretory degradation mediated by the ubiquitin-proteasome pathway and cytosolic hsp70. To further explore the mechanisms of apoB degradation, we have established a cell-free system in which degradation can be faithfully recapitulated. Human apoB48 synthesized in vitro was translocated into microsomes, glycosylated, and ubiquitinylated. Subsequent incubation with rat hepatic cytosol led to proteasome-mediated degradation. To explore whether hsp90 is required for apoB degradation, geldanamycin (GA) was added during the degradation assay. GA increased the recovery of microsomal apoB48 approximately 3-fold and disrupted the interaction between hsp90 and apoB48. Confirming the hsp90 effect in the cell-free system, we also found that transfection of hsp90 cDNA into rat hepatoma cells enhanced apoB48 degradation. Finally, apoB48 degradation was reconstituted in vitro using cytosol prepared from wild type yeast. Notably, degradation was attenuated when apoB48-containing microsomes were incubated with cytosol supplemented with GA or with cytosol prepared from yeast strains with mutations in the homologues of mammalian hsp70 and hsp90. Overall, our data suggest that hsp90 facilitates the interaction between endoplasmic reticulum-associated apoB and components of the proteasomal pathway, perhaps in cooperation with hsp70.