[Mutations in Escherichia coli pmp and htpR genes stabilize the products of foreign gene expression]. / Mutatsii v genakh pmp i htpR Escherichia coli stabiliziruiut produkty ékspressii chuzherodnykh genov.

The half lives of mRNA for Escherichia coli chloramphenicol-acetyltransferase, Bacillus amyloliquefaciens alpha-amylase and human leucocyte interferon were measured in E. coli cells by molecular RNA.DNA hybridization. The effect of mutation in pnp gene, coding polynucleotide phosphorylase, on the stability of these mRNA was studied. The half life of interferon mRNA increases from 25 to 90 s in the pnp mutant, resulting in an increase of interferon accumulation. The stability of interferon in E. coli cells depends on the htpR gene, controlling the heat shock response. The yields of leucocyte interferons alpha-2, alpha I-1 and fibroblast interferon beta increase ten times in htpR mutants. Thus, by using pnp and htpR mutants it is possible to enhance considerably the eukaryotic gene expression in bacterial cells.

[Expression of human leukocyte interferon A gene in Saccharomyces cerevisiae under the control of regulatory yeast elements PHO5, GAL1 and GAL10]. / Ekspressiia gena leikotsitarnogo interferona A cheloveka v drozhzhakh Saccharomyces cerevisiae pod kontrolem reguliatornykh élementov drozhzhevykh genov PHO5, GAL1 i GAL10.

A chromosomal gene for human leucocyte interferon A is expressed in Saccharomyces cerevisiae yeasts due to interaction of 5'-nontranslating region of the cloned interferon gene with the regulatory elements of yeast genes PHO5, GAL1 and GAL10. Regulated systhesis of interferon was obtained in all cases. The level of interferon genes expression in case using GAL1 and GAL10 genes regulatory elements (5 X 10(5) and 5 X 10(6) u X l-1) correlated with the distances to their promoters. The highest yield of interferon (10(8) u X l-1) was obtained when the PHO5 gene regulatory elements were used.

[Expression of the human interferon alpha F gene in the obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida]. / Ekspressiia gena chelovecheskogo interferona alpha F v obligatnom metilotrofe Methylobacillus flagellatum KT i Pseudomonas putida.

The expression of human leucocyte interferon alpha F gene in plasmid pLM-IFN alpha F-273 is controlled by a hybrid tac (trp-lac) promoter. A structural gene for interferon alpha F is a component of the hybrid operon lacZ'-IFN alpha F-TcR, that contains an E. coli trp-operon intercystronic region. Plasmid pLM IFN alpha F-273--directed interferon synthesis allows to obtain about 10(7) IU/l. This plasmid was cloned in broad-host-range vector plasmid pAYC31. The hybrid bi-repliconed plasmid containing interferon gene as well as its single-repliconed deletion derivatives obtained by the in vivo recombination, were introduced into obligate methylotroph Methylobacillus flagellatum KT and Pseudomonas putida PpG6. Methylotrophic strain and Pseudomonas were able to transcribe the interferon gene from E. coli tac promoter, the yield of interferon being 2-4-fold higher as compared with the one in the initial host.

[Expression of human leukocyte interferon type A in Saccharomyces cerevisiae under the control of regulatory elements of the yeast gene URA3]. / Ekspressiia gena leikotsitarnogo interferona A cheloveka v drozhzhakh Saccharomyces cerevisiae pod kontrolem reguliatornykh élementov drozhzhevogo gena URA3.

Expression of a chromosomal gene for human leucocyte interferon A was obtained due to interaction of 5'-nontranslating region of a cloned interferon gene with the regulatory sequences from UNA3 yeast gene. The sequence of 5'-nontranslating region from interferon gene essential for its expression in yeast is localized within 130 b p. from the initiating codon. Due to increasing of plasmid stability and copy number a 60-fold increase. in interferon yield was obtained in yeast transformants reaching the level of 6 X 10(7) u X 1(-1). The data are presented supposing the existence of functional polycistronic mRNA in yeast.

[Isolation and properties of genetic engineered human leukocyte interferons alphaI1 and alphaN]. / Vydelenie i svoistva genno-inzhenernykh chelovecheskikh leikotsitarnykh interferonov alphaI1 i alphaN.

Using controlled pore glass chromatography and immunoaffinity chromatography on monoclonal antibodies NK-2 immobilized on Sepharose 4B, the electrophoretically homogeneous interferons alpha N and alpha I1 were isolated from the biomass of gene-engineered Pseudomonas sp. strains. In terms of specific activity on human fibroblast diploid cells, interferon alpha I1 does not differ from interferon alpha A, whereas the specific antiviral activity of interferon alpha N is as low as 2.10(7) JU/mg. The procedures for immunometric assay of interferons alpha N and alpha I1 have been elaborated. Various monoclonal antibodies to interferon alpha A and to natural leucocyte interferon were analyzed; among those the antibodies specifically interacting with interferons alpha N and alpha I1 (but not with interferon alpha A) were identified.

[Isolation and analysis of human leukocyte interferons synthesized by bacterial producer strains]. / Poluchenie i analiz riada chelovecheskikh leikotsitarnykh interferonov, sinteziruemykh bakterial'nymi shtammami-produtsentami.

Rapid and sensitive methods for immunochemical estimation of bacterially-made human leukocytic interferons were developed. A large number of strains producing such interferons was analyzed with these methods and optimal ones capable of synthesizing up to 10(10), 10(8), 10(9) and 10(9) IU/l of interferons alpha A, alpha N, alpha F and alpha I1 respectively were selected. The proteins were isolated in homogenous state and their main physico-chemical parameters were characterized. Interferon oligomers were detected. Their formation was partially due to intermolecular disulphide exchange. The effect on interferon alpha A of retinal (vitamin A) and its derivatives efficiently interacting with this protein was studied.

[Comparative immunochemical analysis of various human leukocyte interferons]. / Sravnitel'nyi immunokhimicheskii analiz nekotorykh leikotsitarnykh interferonov cheloveka.

Using radioimmunological methods based on the use of mono- and polyclonal antibodies raised against interferon alpha A, it was shown that polyclonal antibodies quantitatively reacted not only with this protein, but also with interferons alpha F and alpha N, whereas all the variants of monoclonal antibodies studied reacted only with interferons alpha A and alpha N. Monoclonal antibodies 5A6, 11E9, 19C10, 258 and 268 are directed against overlapping epitopes of the interferon alpha A molecule, which simultaneously binds not more than two molecules of antibodies with different specificity. The correlation between immunochemical and biological activities of interferon alpha A during temperature denaturation and proteolytic degradation and its ability to form oligomeric complexes were investigated.