摘要
按本室纯化的N 氨甲酰基 D 氨基酸酰胺水解酶 (DCase)的N 末端氨基酸序列设计了正向引物 ,并在已报道的不同来源菌株DCase的氨基酸序列同源性分析的基础上 ,设计了反向引物。用菌株No . 2 2 62总DNA为模板 ,经PCR扩增获得长度为约0 9kpb的片段 ,DNA序列分析表明基因全长为 912bp。将该片段插入 pET 3a ,构建成重组子 pET DCase ,并在E coliDH5α中获得表达 ,经SDS PAGE检测 ,表达产物分子量与天然纯DCase相同 ,约为 35kD。
D-Carbamoylase(DCase) gene was amplified from genomic DNA of strain No.2262 by PCR, using the forward primer deduced by N-terminal amino acid sequence of DCase purified by this Lab. and the reverse primer designed by C-terminal amino acid sequence of DCase according to the amino acid sequence homology of some strains. PCR product shows about 0.9kb fragment on agarose gel. Then the DNA fragment was inserted into vector pET-3a to construct a recombinant, pET-DCase. It was shown from DNA sequence analysis that the open reading frame of DCase is 912bp corresponding 304 amino acid residues. When the engineered strain E.coli DH5α/pET-DCase was expressed in LB+Amp medium by the induction of IPTG ,a band with MW of 35kD which was similar to pure DCase was occurred by SDS-PAGE . The results showed from all of the experiments described above that the expression product could be DCase. In addition, the homology of amino acid sequence deduced from DNA sequence of the enzyme is also compared to that from some reported strains.
出处
《高技术通讯》
EI
CAS
CSCD
2003年第1期32-36,共5页
Chinese High Technology Letters
基金
山西省重点科技发展项目 ( 9932 2 5 )
