Cloning and expression of botulinum neurotoxin type B light chain region and characterization of its recombinant protein = 보튜리눔 신경독 B형 Light chain의 클로닝과 발현 및 재조합 단백질의 특성에 관한 연구

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It was cloned and sequenced light chain region of botulinum neurotoxin type B which is potent neurotoxin produced by Clostridium botulinum type B Lamanna. From amino acid sequences deduced from nucleic acid sequences, it was known that light chain of botulinum type B neurotoxin is composed of 441 codons and molecular mass is 58,827.5 Da. When the amino acid sequences of light chain was compared with that of other types of clostridial neurotoxin, BoNT/B has high similarity with BoNT/G and TeNT (61 % and 52 % similarity, respectively). These indicate that BoNT/B, BoNT/G and TeNT have a high genealogical relatedness. Recombinant light chain was expressed and purified from E. coli with relatively simple chromatography using two ion exchange resin, DEAE-cellulose and phosphocellulose. The purified light chain actively cleaved the synaptobrevin of the mouse brain microsome. But when treated with metal chealating agent (EDTA or 2,2``-dipyridyl), the activity was inhibited. It was confirmed that the purified protein was light chain and was not contaminated with other protein with anti- recombinant light chain antibody obtained from rat. Native BoNT/B was also purified with APTG affinity chromatography which interacts with haemagglutinin moieties of neurotoxin complex and DEAE cellulose ion exchange chromatography. When the activity of the recombinant light chain was compared with native BoNT/B in dose and time dependent manner, recombinant light chain have activity as same as that of native light chain. The activity of light chain was analyzed in various pH and salt concentration. The catalytic activity of the recombinant light chain was maintained stable from pH 3 to pH 9 but drastically decreased below pH 3.0 and above pH 9.0. The activity of light chain rapidly decreased in higher concentration than 0.2 M NaCl.
Yang, Kyu-Hwan양규환
한국과학기술원 : 생물과학과,
Issue Date
112588/325007 / 000895348

학위논문(박사) - 한국과학기술원 : 생물과학과, 1997.2, [ x, 110 p. ]


Cloning; Expression; Activity; 보듀리눔; 신경독; 클로닝; 발현; 반응성; Botulinum; Neurotoxin

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