[Recombinant human insulin: X. Simplification of folding of the biotechnological precursor of recombinant human insulin].

The folding of biotechnological precursor of human insulin precursor was carried out from solubilized inclusion bodies without a preliminary oxidizing or reducing of Cys residues. The inclusion bodies were dissolved in 8 M urea with addition of 10 mM 2-mercaptoethanol. Hydrophobic cell components were removed from the solution by passing through a neutral weakly hydrophobic sorbent, the solution was five times diluted and refolded upon addition of 0.3 mM cystine for initiation of disulfide rearrangement. The presence of nucleic acids and cell protein impurities does not affect the folding efficiency. The resulting precursor of folded human insulin was purified by metal-chelate affinity chromatography and converted into insulin by two-stage enzymatic cleavage.

[Methods of preparation of recombinant proteins-cytokines. IV. Renaturation of recombinant human interleukin-3]. / Metody polucheniia recombinantnykh belkov-tsitokinov IV. renaturatsiia rekombinantnogo chelovecheskogo interleikina-3.

Renaturation of recombinant human interleukin-3 produced as inclusion bodies in the transformed cells of Escherichia coli was studied and optimized. Importance was shown of removing from the protein solution the hydrophobic cellular components causing irreversible aggregation of the protein under renaturation conditions. An effect of pH on the secondary structure of the denatured protein was revealed by CD spectroscopy. It was thereby found that at pH 8.5, which is the optimal value for denaturation, the protein has the secondary structure most close to the native one. The isolation according to the scheme proposed allows preparation of interleukin-3 in 50% yield with 99% purity and biological activity 2 x 10(7) U/mg.

[Recombinant human insulin. VII. Increased efficacy of chromatographic separation based on a principle of bifunctionality]. / Genno-inzhenernyi insulin cheloveka. VII. Povyshenie éffektivnosti khromatograficheskogo razdeleniia pri ispol'zovanii printsipa bifunktsional'nosti.

Retention mechanisms of insulin and deamido[AsnA21] insulin on the bifunctional sorbent Armsphere-C8(PR) in conditions of reversed-phase chromatography (HPLC and ion-pair HPLC) were studied. In accordance with the chemical differences of these proteins, molecular mechanisms of their interaction with silica gel modified with hydrophobic and ion-exchange groups were revealed. The possibility of simultaneous interaction of sorbed proteins with the stationary phase by both mechanisms under conditions of reversed-phase HPLC was demonstrated. The dependences of the separation selectivity and resolution on the mobile phase composition and properties (a salt buffer type, pH, ionic strength) were found. It was demonstrated that the separation selectivity can be regulated by altering the contribution of each of the two separation mechanisms and the bifunctional sorbent used allows higher selectivity in the separation of close protein analogs than monofunctional sorbents.

[Preparation of recombinant cytokines. II. Effective method for isolation, purification and renaturation of human recombinant gamma-interferon]. / Metoddy polucheniya rekombinantnykh belkov-tsitokinov. II*. 'Effektivnyl metod vydeleniya irenaturatsii rekombinantrnogo gamma-interferona cheloveka

An efficient method for the isolation, purification, and renaturation of human recombinant gamma-interferon from biomass of transformed E. coli cells was developed. It involves the extraction of the protein from the inclusion bodies, preliminary purification of the protein, and three stages of ion-exchange chromatography with an intermediate renaturation between the second and the third stages. A highly active (2 x 10(7) U/mg) recombinant protein of up to 99% purity (according to SDS-PAGE and HPLC) was obtained with a 30% overall yield.

[An effective method of purifying recombinant human tumor necrosis factor alpha]. / Effektivnyi metod ochistki rekombinantnogo alpha-faktora nekroza opukholei cheloveka.

An efficient and productive isolation method for human recombinant tumor necrosis factor alpha from Escherichia coli cells was developed. The method includes a membrane filtration step, two steps of ion-exchange chromatography, and gel filtration on a Sephadex G-25 column. The target product was obtained with approximately 50% total yield and greater than 95% purity according to PAGE and HPLC.

[Effect of the topography of the signal peptidase site on the effectiveness of secretion of recombinant human granulocyte-macrophage colony-stimulating factor into Escherichia coli periplasm]. / Vliianie topografii saita signal'noi peptidazy na éffektivnost' sekretsii v periplazmu Escherichia coli rekombinantnogo granulotsitarno-makrofagal'nogo koloniestimuliruiushchego faktora cheloveka.

Synthesis of an artificial gene encoding the signal peptide of the Yersinia pestis capsule antigen (Caf1) was accomplished. A set of plasmids coding for hybrid proteins in which a modified sequence of the Caf1 signal peptide is connected to the amino acid sequence of the mature granulocyte-macrophage colony stimulating factor (GM-CSF) were constructed. Topography of the cleavage site of signal proteases was studied. The presence of an arginine residue within the N-terminal part of the mature human GM-CSF was shown to hinder the proper processing and translocation of the precursor through periplasmic membrane. A number of E. coli strains secreting biologically active mutants of human GM-CSF were obtained.

[Genetic engineering of human insulin. IV. Development and optimization of an analysis system using reversed phase high pressure liquid chromatography]. / Genno-inzhenernyi insulin cheloveka. IV. Razrabotka i optimizatsiia sistemy analiza s pomoshch'iu obrashchenno-fazovoi vysokoéffektivnoi zhidkostnoi khromatografii.

The effectiveness of the RP HPLC application for the step-by-step analysis of the recombinant insulin production was studied. Properties of a number of commercial and experimental columns in different chromatographic conditions were considered. A three-dimension optimization of selectivity and resolution versus pH and ion strength was carried out. A mechanism of the resolution and selectivity control is suggested.

[Separation of recombinant proteins by chromatography on vesicular sorbents]. / Razdelenie rekombinantnykh belkov s pomoshch'iu khromatografii na vezikuliarnykh sorbentov.

Vesicle chromatography, a recently developed method for separation of biomolecules, uses the vesicular packing (VP) material (clusters of microcapsules derived from plant cells), which was tested with respect to its application for the recombinant protein separation. Since VP has a well-defined separation limit, biomolecules are distributed in two separate peaks: large molecules are excluded and small molecules permeate through cell walls into the empty cell lumen. Recombinant proteins frequently form oligomers, which differ from monomers not only in size but also chemically and biologically. In the present study, separations of the recombinant proinsulin fusion protein oligomer and monomer, the recombinant human gamma-interferon monomer and dimer and recombinant tumour necrosis factor-alpha were investigated. For peak identification, the fractions and starting samples of the recombinant proteins were analysed by HPLC. The separations occurred without any sorption effects and with high efficiency and resolution of the protein peaks at a short column (10 cm). The VP is characterised by a high load ability, which favours the scale-up purification of the recombinant proteins. The combination of VP and HPLC is a considerable advance in biotechnology separation.

[Genetically engineered human insulin. 1. HPLC in analyzing products of basic production stages]. / Genno-inzhenernyi insulin cheloveka. 1. VEZhKh v analize produktov osnovnykh stadii proizvodstva.

Application of some variants of HPLC for the step-by-step analysis of recombinant human insulin production was studied. Chromatographic columns with commercial and specially developed supports for size-exclusion, ion-exchange and reverse phase HPLC were used. Effective combinations of the chromatographic techniques for analysis of products and intermediates at every technological step were found and used for production of insulin. The authenticity of insulin obtained in the Shemyakin Institute of Bio-organic Chemistry by the scheme described in the present paper was confirmed by means of some physical and chemical methods and biological activity analysis.

[High performance liquid chromatography of nucleotides. Major methods and their development]. / Vysokoéffektivnaia zhidkostnaia khromatografiia nukleotidov. Osnovnye metody i ikh razvitie.

The separation of mono- and oligonucleotides possibilities by means of high performance ion-exchange, reversed-phase, so-called "ion-pair" and adsorption chromatography are studied. The influence of the eluent composition (solvent, salt) and pH on the retention, selectivity and resolution in reversed-phase and ion-exchange chromatography is investigated. The model of the hydrophobic-pair ion-exchange mechanism of ion-pair chromatography is considered. The conditions for analysis and preparative isolation of a desired component are optimized for selectivity, resolution and throughput. The methods for prediction of the optimal gradient elution program reasonable resolution at the desired retention time and for choosing the guard-column packing material are proposed. A design of the gradient for system and the version of slurry packing method for HPLC prolonged life-time columns are improved. The automatized analytical technique for determination of the oligonucleotide monomeric composition with two coupled microcolumns is described, that involves enzymatic digestion of an oligonucleotide followed by ion-exchange separation of the hydrolysate.