[Extracellular production of recombinant human epidermal growth factor (hEGF) in Escherichia coli cells]. / Ekstrakletochnaia produktsiia rekombinantnogo épidermal'nogo faktora rosta cheloveka (hEGF) v kletkakh Escherichia coli.

An expression plasmid pPTK-hEGF2 was constructed to provide for the extracellular production of recombinant human epidermal growth factor by the Escherichia coli cells. The plasmid contained two expression cassettes, one of which carried a tandem of the fused genes ompF-hegf under the control of the tac promoter, ensuring regulated secretion of hEGF into the E. coli periplasm, and another one contained the kil gene from the ColE1 plasmid under the control of lac promoter. The regulated low-level biosynthesis of Kil protein increased the permeability of E. coli outer membrane for periplasmic proteins. This enabled the recombinant proteins secreted into the cell periplasm to outflow into the cultural medium. As a result, the E. coli strains that harboured this plasmid construct produced effectively the recombinant hEGF into the cultural medium. The yields of hEGF produced by the nTG1(pPTK-hEGF2) and HB101(pPTK-hEGF2) strains reached 25 and 30 mg/l of cell culture after 14 and 18 h of cultivation, respectively. The hEGF preparation isolated possessed biological activity both in vivo and in vitro.

[Expression of synthetic human interleukin-10 gene and its mutant variants in Escherichia coli cells]. / Ekspressiia cinteticheskogo gena interleikina-10 cheloveka i ego variantov v kletkakx Escherichia coli.

The human interleukin-10 gene was obtained by chemico-enzymatic synthesis, and vectors for cytoplasmic and periplasmic expression of the recombinant IL-10 gene in Escherichia coli cells were constructed. Mutant IL-10 genes bearing substitutions in a region upstream of the ATG codon and in the triplet coding for the second amino acid residue in the protein were obtained by in vitro mutagenesis. High levels of expression were observed for the fusion protein composed of IL-10 and an N-terminal fragment of IL-3 and for the mutant IL-10 containing cysteine as the second amino acid residue.

[Recombinant interleukin-3 expression system in E. coli]. / Sistemy ékspressii v E. coli rekombinantnogo interleikina-3.

Plasmid pTOTE2IL3 (III) has been constructed, expressing an artificial human interleukin-3 (hIL3) gene under conditions of the induced protein biosynthesis. Levels of the recombinant protein synthesis have been compared in several E. coli strains containing expression plasmids pTE2IL3 (I) (constitutive biosynthesis) and (III) (induced biosynthesis). Optimal combinations of the expression plasmids and the bacterial strains are of importance. A simple and effective method has been elaborated for isolation, purification and renaturation of the recombinant protein accumulated in inclusion bodies.

[Production of recombinant human keratinocyte growth factor in Escherichia coli cells]. / Produktsiia rekombinantnogo faktora rosta keratinotsitov cheloveka v kletkakh Escherichia coli.

Here we describe the synthesis of the gene encoding human keratinocyte growth factor (hKGF) and the construction of vectors for cytoplasmic production of hKGF in Escherichia coli cells. The level of recombinant protein expression was 1-1.5% of the total cell protein. hKGF was purified to homogeneity by three-step ion-exchange and affinity chromatography. The protein is biologically active: it stimulates dose-dependent protein synthesis in cultured human epidermal keratinocytes.

[Production of recombinant hIL-4delta2-a native isoform of human interleukin-4 in Escherichia coli cells]. / Produktsiia rekombinantnogo hIL-4delta2-prirodnoi izoformy interleikina-4 cheloveka, v kletkakh Esherichia coli.

Expression plasmids containing the synthetic gene hil-4 delta 2 was constructed to produce human interleukin-4 in Escherichia coli cells. Strains TG1 (pBTIL-4 delta 2) and BL21 (DE3) (pETIL-4 delta 2) produced the recombinant protein as inclusion bodies, and its production level was up to 30% of the total cell protein. The renatured hIL-4 delta 2 inhibited IL-4-stimulated T cell proliferation, and this effect was enhanced by cyclosporin A.

[Expression of Photobacterium leiognathi bioluminescence system genes in Escherichia coli]. / Ekspressiia genov bioliuminestsentnoi sistemy Photobacterium leiognathi v Escherichia coli.

Expression of Photobacterium leiognathi bioluminescence genes under the control of lac, tac, tet promoters in Escherichia coli cells has been studied. The position of the genes for aliphatic aldehyde biosynthesis and for the synthesis of luciferase subunits was identified. The plasmid pBRPL1 has been constructed containing the system of bioluminescence genes devoid of promoter following the polylinker DNA fragment. The plasmid can be used for selection of promoter containing DNA sequences as well as for studying the promoters regulation in process of Escherichia coli cells growth.

[Cloning and insertion mutagenesis of DNA fragment coding for the luminescent system of Photobacterium leiognathi]. / Klonirovanie i insertsionnyi mutagenez fragmenta DNK, kodiruiushchego liuminestsentnuiu sistemu Photobacterium leiognathi.

Fragments of DNA, obtained from the luminescent bacterium Photobacterium leiognathi and inserted into the plasmid pBR322, were found to code for the luminescence expressed in E. coli cells. The genetic functions necessary for light production in E. coli are localized on a DNA fragment of about 7 kbp. The insertion mutagenesis was used to define the luminescence functions encoded by the hybrid plasmid.

[Use of Escherichia coli recF mutant strains for analyzing the genotoxicity of complex platinum compounds]. / Primenenie redF mutantnykh shtammov Escherichia coli dlia analiza genotoksichnosti kompleksnykh soedinenii platiny.

The bioluminescence method of assessing SOS response in Escherichia coli cells was applied to test the genotoxicity of five complex platinum compounds: cis-diamminedichloroplatinum, cycloplatam, trans-diamminedichloroplatinum, and two trans-binuclear complexes. Strains AB1157 (pPLS-1) and JC9239 (pPLS-1), a variant of AB1157 carrying the recF143 mutation, were used in the test procedure. SOS response in JC9239 cells was shown to be induced by significantly lower concentrations of cis-DDP and cycloplatam than in AB1157 cells. A drastic increase in cis-DDP toxicity for the JC9239 strain was shown. However, the presence of the recF mutation in JC9239 cells retarded the SOS response induced by trans-platinum compounds. The results obtained indicated that the E. coli recF system plays an essential role in repair and utilization of cis-DDP-DNA adducts.

[UV-light-tested protein--DNA interactions in the nuclei during the cell cycle in Physarum polycephalum]. / Testiruemye uf-svetom belok--DNK-kontakty v iadrakh na raznykh stadiiakh kletochnogo tsikla Physarum polysephalum.

The release of DNAs from nuclear preparations isolated at different steps of the cell cycle of the synchronous culture of Physarum polycephalum and irradiated with UV-light for 5 min (lambda 253.7 A, i = 1.8 . 10(2) j . m-2 . min-1) into 1% Na-DS - 1.5 M NaCl solution was studied. The value obtained correlated well with the degree of binding of interacting proteins with DNA in chromatin and was identical for all the preparations tested with the only exception of the nuclei isolated at the end of the S-phase, when a slight but significant (15 +/- 10%) increase in DNA release was observed. The results obtained are discussed in terms of structural transitions and function of chromatin during the cell cycle of the myxomycete Physarum polycephalum.

[Separation and analysis of immunochemical properties of multiple forms of cytochrome P-450 from the rat liver]. / Razdelenie i issledovanie immunokhimicheskikh svoistv mnozhestvennykh form tsitokhroma P-450 pecheni krys.

Four cytochromes P-450 induced by phenobarbital (PB-1--PB-4) and two cytochromes P-450 induced by S-methylcholanthrene (MC-1, MC-2) were purified to electrophoretic homogeneity from rat liver microsomes. The purification procedure involved sequential chromatography on n-aminooctyl-Sepharose 4B, DEAE-Sephacel and hydroxylapatite columns. The spectral and immunochemical properties of the cytochromes P-450 were estimated. All, but MC-1, cytochromes P-450 were found to exist in a low spin state. Using the Ouchterlony double diffusion method, it was shown that all cytochromes P-450 under study can be divided into two groups, i. e., PB-1--PB-2 and PB-3--PB-4, sharing common antigenic determinants inside the groups. High performance liquid chromatography of PB-3 and MC-2 on anion-exchangers yielded two additional peaks from the PB-induced major cytochrome P-450 PB-3 and three peaks from the MC-induced major cytochrome P-450 MC-2. The multiplicity of cytochrome P-450 forms is discussed.

A biosensor for environmental genotoxin screening based on an SOS lux assay in recombinant Escherichia coli cells.

A genetically controlled luminescent bacterial reporter assay, the SOS lux test, was developed for rapid detection of environmental genotoxins. The bioassay is based on the recombinant plasmid pPLS-1, which was constructed as a derivative of pBR322, carrying the promoterless luxCDABFE genes of Photobacterium leiognathi downstream of a truncated cda gene from ColD with a strong SOS promoter. E. coli recA+ strains containing this construction are inducible to high levels of light production in the presence of substances or agents that cause damage to the DNA of the cells. The light signal, reflecting the SOS-inducing potency, is recorded from the growing culture within 1 s, and the test results are available within 1 to 2 h. Induction of bioluminescence was demonstrated by treatment of E. coli C600(pPLS-1) with 6 genotoxic chemicals (mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, nalidixic acid, dimethylsulfate, hydrogen peroxide, and formaldehyde) and with UV and gamma radiation. A clear dose-response relationship was established for all eight genotoxins. The sensitivity of the SOS lux test is similar to that of other bioassays for genotoxicity or mutagenicity, such as the SOS chromotest, umu test, and Ames mutatest. These results indicate that the SOS lux test is potentially useful for the in situ and continuous detection of genotoxins.

[Effect of UV-light on the structure of soluble deoxyribonucleoprotein-200 A]. / Vliianie uf-sveta na struktupu rastvorimogo dezoksiribonukleoproteida 200 A.

The effects of UV-light (253,7 nm) on the structure of DNP and its protein and nucleic components were studied. The formation of protein-DNA covalent bonds in DNP-200 A at low ionic strength was confirmed. Under certain irradiation conditions more than 80% of protein may be linked to the DNA; all histone fractions were linked to the same extent and at the same rates. The local denaturation of DNA in the region of photo-induced thymine-thymine dimers and other photoadducts dramatically changed the rate and specificity of the effects of staphylococcal nuclease, which directly affected the composition and size of the fragments formed. A possible application of UV-irradiated DNP for various structural investigations is discussed.