[Protein engineering of uridine phosphorylase from Escherichia coli K-12. II. Comparative study of hybrid and mutant forms of uridine phosphorylases]. / Belkovaia inzheneriia uridinfosforilazy Escherichia coli K-12 II. Sravnitel'noe izuchenie svoistv gibridnykh i mutantnykh form uridinfosforilaz.

Genes for hybrid uridine phosphorylases (UPases) consisting of fragments of amino acid sequences of UPases from Escherichia coli and Salmonella typhimurium were constructed. Producing strains of the corresponding proteins were genetically engineered. Mutant forms of the E. coli K-12 UPase were produced by site-directed mutagenesis. A comparative study of the enzyme properties of the mutant and hybrid forms of bacterial UPases was performed. It was shown that Asp27 unlike Asp5 and Asp29 residues of the E. coli UPase forms part of the active site of the protein. A scheme of the involvement of Asp27 in the binding of inorganic phosphate is proposed.

[Design of the human tumor-associated VNTR(Muc1) antigen gene, fused with streptavidin, its expression in Escherichia coli. Study of properties of the hybrid protein]. / Konstruirovanie gena opukholeassotsiirovannogo antigena VNTR(Muc1) cheloveka, slitogo so streptavidinom, ego ékspressiia v Escherichia coli. Izuchenie svoistv gibridnogo belka.

A gene of human tumor-associated antigen VNTR(MUC1) bound to streptavidin, an expression plasmid, and a highly effective hybrid protein-producing strain were constructed. It was shown that the streptavidin leader peptide ensures an effective secretion of the hybrid protein into the periplasmic space of Escherichia coli cells. The hybrid protein was isolated in a homogeneous state and its immunogenic properties were studied.

[Cloning of streptavidin gene from Streptomyces avidinii and its expression in Escherichia coli. Secretion of streptavidin by E. coli cells]. / Klonirovanie gena streptavidina iz Streptomyces avidinii i ego ékspressiia v Escherichia coli. Sekretsiia streptavidina kletkami E. coli.

The streptavidin gene from Streptomyces avidinii was cloned, an expression plasmid constructed, and a highly effective strain producer of streptavidin created. It was shown that the leader peptide of streptavidin ensures the effective secretion of this protein into the periplasmic space of Escherichia coli cells. The degradation site of the leader peptide was detected. Upon treatment with the total fraction of proteases secreted by S. avidinii into the culture medium, "core" streptavidin was obtained, which retained the biotin-binding function.

[Protein engineering of uridine phosphorylase from Escherichia coli K-12. I. Cloning and expression of uridine phosphorylase genes from Klebsiella aerogenes and Salmonella typhimurium in E. coli]. / Belkovaia inzheneriia uridinofosforilazy iz Escherichia coli K-12. I. Klonirovanie i ékspressiia v E. coli genov uridinofosforilaz iz Klebsiella aerogenes i Salmonella typhimurium.

Genes of uridine phosphorylases (udp) from Klebsiella aerogenes and Salmonella typhimurium were cloned and expressed. Highly effective producer strains of the corresponding proteins were constructed. Enzymic properties of the UPases obtained were studied and compared with those from the Escherichia coli enzyme. Mutant forms of UPase from E. coli (D5E, D5N, D5A) were prepared by site-directed mutagenesis techniques. It was shown that the Asp5 residue plays an insignificant role in the formation of the active form of the protein.

[Cloning transforming genes from human papillomavirus type 18]. / Klonirovane transformiruiushchikh genov virusa papillom cheloveka tipa 18.

Nucleotide sequences of type 18 human papilloma virus genes E6 and E7 inserted in human DNA cloned from cervical tumor are determined. The resultant sequences are compared to the prototype variant. Five point mutations not leading to replacement of amino acid residues in polypeptide chain and 1 substituting the amino acid in codon 129 are detected in gene E6 sequence. In E7 sequence, one significant mutation in codon 92 is detected. Both substitutions of asparagine for lysine are localized in the 3'-terminal region of the genes, which may not affect the transforming potential of these sequences. DNA fragments of E6 and E7 coding regions obtained by PCR were independently cloned in bifunctional expression vector DelpC7. The identity of hybrid E6 and E7 nucleotide sequences to initial ones is verified by sequencing.

[Study of the role of histidine residues in the function of uridine phosphorylase from Escherichia coli K-12 by protein engineering]. / Issledovanie roli ostatkov gistidina v funktsionirovanii uridinfosforilazy iz Escherichia coli K-12 metodami belkovoi inzhenerii.

Using site-directed mutagenesis, mutant genes of the E.coli UDPase that coded proteins with point substitutions of histidine residues (i.e., H8N, H47N, H101N, H122N, H152N, H179N, and H240N) were constructed. Study of the enzymatic activity of mutant UDPases showed that histidine-asparagine substitutions at the positions 47, 101, 152, 179, and 240 do not affect protein functioning. Whereas H122N and H8N substitutions inhibit the activity of UDPase by 60 and 100%, respectively. This evidences the important functional role of the His122 and His8 residues for the formation of the active site fo the enzyme.

[Chemical synthesis of a proteinase inhibitor (eglin C) gene without use of T4-DNA-ligase and its expression in E. coli]. / Khimicheskii sintez gena ingibitora proteinaz (églin C) bez ispol'zovaniia T4-DNK-ligazy i ego ékspressiia v E. coli.

A synthetic gene for a proteinase inhibitor (eglin C) that was obtained by direct amplification with oligonucleotides without using DNA ligase and polynucleotide kinase of T4 phage was cloned into expression vectors. A high yield of the polypeptide (110-130 mg/l) was attained in E. coli strains.