Biomedical subjects
Publications and source records attributed to A F Sheval'e.
The possible use of MALDI TOF MS for the analysis of Escherichia coli strains producing recombinant proteins was studied. It was shown that the target chimerical proteins might be rapidly detected in the strains.
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Three peptides corresponding to the sequences 124-144, 124-138, 129-144 of the human leukocyte interferon alpha 2 (IFN-alpha 2) were synthesized. The synthesis was performed by DCC-HOBT coupling of protected peptide segments in solution. The segments were obtained by the active ester coupling methodology using base-labile 2-[4-(phenylazobenzyl)sulfonyl]ethyl (Pse) group as carboxyterminal protection. After complete deprotection with 1 M methanesulphonic acid in trifluoroacetic acid--thioanisol--m-cresol mixture the peptides were purified by reversed-phase chromatography. The studies of interaction of the peptides with rabbit antiserum against IFN-alpha 2 revealed at least one minor antigenic determinant within the 124-144 region of IFN-alpha 2 amino acid sequence. Rabbit antisera developed against peptides 124-138 and 129-144 showed ability of binding recombinant IFN-alpha 2 and neutralizing its antiviral activity. Free peptides or their conjugates with bovine serum albumine did not display antiviral activity, neither could they inhibit the activity of IFN-alpha 2.
A sensitive nonradioactive method for detection of substances interacting with the neuronal nicotinic acetylcholine alpha 7-type receptor (AChR) was proposed. The method uses biotinylated alpha-cobratoxin (Bt-CTX) and is based on the ability of the N-terminal ligand-binding extracellular domain (LBED) of AChR to interact with alpha-cobratoxin (CTX) as does the whole receptor. LBED was produced by heterologic expression of a gene fragment of the alpha 7 subunit of AChR from the rat brain in Escherichia coli cells sorbed on wells of a 96-well plate and incubated with Bt-CTX. The specifically bound Bt-CTX was determined by staining with streptavidin-peroxidase complex. The ability of other compounds to interact with alpha 7-AChR was checked according to the degree with which they inhibit Bt-CTX binding to LBED. Nicotine, carbamylcholine, d-tubocurarin, anabaseine, conotoxin ImI, and neurotoxin II were used as model compounds. The sensitivity of this method was comparable with that of the radioligand method (up to 10 pmol).