Russian Journal of Bioorganic Chemistry, Vol. 21, No. 1, 1995, p. 68–69

Rapid Detection of Point Mutations with Biotinylated Selectivity Cleavable Synthetic Oligonucleotides

M. S. Shchepinov, V. G. Korobko, V. N. Dobrynin, F. A. Amosenko ^* , and V.N.Kalinin ^*

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, V-437, GSP-7, Moscow , 117871 Russia

^* Institute of Human Genetics, Russian Academy of Medicinal Sciences, Moscow

Abstract: The method is described for fast detection of point mutations using originally prepared 5’-biotinylated specifically chemically cleavable oligonucleotides by means of allele-specific amplification in the presence of [³³P]TTP, immobilization of the products to streptavidinylated support and subsequent successive splintering of cleavable inserts.

Key words: point mutations, polymorphism, cystosic fybrosis, allele-specific primers, biotin, selectively cleavable primers.

Russian Journal of Bioorganic Chemistry, Vol. 21, No. 6, 1995, p. 370–374

Cloning cDNA Encoding F _v -Fragments of the Light and Heavy Chains of the Monoclonal Antibody against Human Interleukin-2

A.I. Paskhin ^* , T.N. Golovina, V.A. Nesmeyanov, and V.G. Korobko

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, V-437, GSP-7, Moscow , 117871 Russia

Abstract: DNA fragments coding for the variable fragments of the heavy and light chains of the immunoglobulin G₁ against human recombinant interleukin-2 were produced using reverse transcription of the total RNA isolated from the murine hybrid myeloma cell line LNKB-2 followed by amplification of the RNA-DNA duplexes with degenerated primers. The fragments obtained were cloned into the plasmid pGEM7-Zf(+) and their structures were determined. The fragments cloned were proved to encode the F_v-fragments by sequencing N-termini of the light and heavy chains of the antibody.

Key words: immunoglobulin G ₁ , F _v -fragments, cloning in E.coli, cDNA, reverse transcription8

Russian Journal of Bioorganic Chemistry, Vol. 21, No. 7, 1995, p. 452–454

Cloning and Highly Efficient Expression of the Gene Encoding Escherichia coli Thioredoxin

M.G.Barenboim, L.N.Shingarova, and V.G. Korobko

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, V-437, GSP-7, Moscow , 117871 Russia

Abstract: The gene encoding thioredoxin (trxA) was isolated from chromosomal DNA of E. coli HB101 strain using the polymerase chain reaction. The cloned structural gene with a synthetic Shine-Dalgarno sequence was placed under the control of either inducible tac-promoter or a tandem of two strong constitutive promoters A2 and A3 from early region of bacteriophage T7. Both constructions were shown to provide high levels of biosynthesis of recombinant thioredoxin.

Key words: thioredoxin, expression in E.coli, polymerase chain reaction

Russian Journal of Bioorganic Chemistry, Vol. 21, No. 11, 1995, p. 731–738

Construction of Recombinant E. coli Strains for Secretory Expression of Artificial Genes for Human Granulocyte-Macrophage Colony Stimulating Factor

L.E. Petrovskaya, A.V. Ruzin, L.N.Shingarova, and V.G. Korobko

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, V-437, GSP-7, Moscow , 117871 Russia

Abstract: A number of recombinant plasmids for expression of artificial genes encoding human granulocyte-macrophage colony stimulating factor (GM-CSF) were constructed. A hybrid gene was obtained that contains a sequence encoding the leader peptide and a tandem of two IgG-binding domains of protein A from Staphylococcus aureus coupled, through an enteropeptidase linker, to a synthetic gmcsf gene. The construction enables Escherichia coli to carry out biosynthesis of the hybrid protein and its subsequent transport into the periplasmic space of bacteria. Another hybrid gene, combining sequence for the signal peptide of the E.coli outer membrane protein OmpA and GM-CSF, was obtained using polymerase chain reaction. The localization of the mature protein produced by the hybrid gene was found to depend on the strength of the promoter use.

Key words: granulocyte-macrophage colony stimulating factor (GM-CSF), gene, expression in E.coli, secretion, polymerase chain reaction

Russian Journal of Bioorganic Chemistry, Vol. 21, No. 12, 1995, p. 785–791

Effect of Topography of the Signal Protease Cleavage Site on the Efficiency of Secretion of Human Granulocyte-Macrophage Colony Stimulating Factor into Periplasm of Escherichia coli Cell

L.E. Petrovskaya, E.A. Krukova, S.A. Yakimova, A.N. Wolfson, R.A. Alibaeva, L.N.Shingarova, A.A. Guzaev ^* , V.M. Abramov*, and V.G. Korobko

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, V-437, GSP-7, Moscow, 117871 Russia, ^* Institute of Immunoengineering , Lyubuchany, Moscow oblst 142380 Russia

Abstract: 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 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.

Key words: granulocyte-macrophage colony stimulating factor (GM-CSF), hybrid gene, capsule antigen F1 (Caf1), signal sequence, expression in E.coli, secretion