重组赖氨酸内肽酶构建表达及活性方法研究  

Research of Recombinant Lysine Endopeptidase Construction Expression and Activity

作　　者：马妍[1] 付志成 范开[1] 

机构地区：[1]重庆理工大学药学与生物工程学院,重庆400054 [2]富进生物医药有限公司,重庆400041

出　　处：《重庆理工大学学报（自然科学）》2015年第4期53-59,106,共8页Journal of Chongqing University of Technology：Natural Science

摘　　要：设计了重组赖氨酸内肽酶蛋白氨基酸序列,根据P.Pastoris酵母密码子偏爱性原则设计c DNA序列,其中TGA和TAA为两个终止密码子序列,并在序列5’端和3’端分别设计Xho I(CTCGAG)和Not I(GCGGCCGC)限制性酶切位点,将其重组到原核表达质粒p MD19-T中,得到p MD19-T-Lys-C,并在毕赤酵母中获得表达。应用SDS-PAGE电泳及免疫斑点法检测其表达,RP-HPLC分析重组赖氨酸内肽酶可以特异性切除赖氨酸基团C末端的氨基酸残基,证明该重组的赖氨酸内肽酶可替代Lysyl Endopeptidase应用于胰岛素的制备工艺中。The amino acid sequence of lysine peptide endopeptidase in peptide was recombined and c DNA sequence was designed according to P. Pastoris yeast codon preference principle,including two termination codons which were TGA and TAA. The restriction sites,Xho I（ CTCGAG） and Not I（ GCGGCCGC） were designed at 5＇end and 3＇end of c DNA sequence. The sequence was recombined to prokaryotic expression plasmid p MD19-T,and p MD19-T-Lys-C was obtained and expression was acquired in pichia pastoris. SDS-PAGE and dot immunobinding assay were used in the determination of expression. RP-HPLC analysis proved that amino acid residue at C end of lysine groups can be excised specifically by lysine peptide endopeptidase recombination. Lysine peptide endopeptidase recombination can replace Lysyl Endopeptidase in the preparation technology of insulin.

关 键 词：基因重组 表达 胰岛素原 活性 

分 类 号：R394[医药卫生—医学遗传学]