CAJ | 학술논문

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 cDNA 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 pMD19-T中，得到 pMD19-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-GPLC 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。
설계료중조뢰안산내태매단백안기산서렬，근거 P. Pastoris 효모밀마자편애성원칙설계 cDNA 서렬，기중 TGA 화 TAA 위량개종지밀마자서렬，병재서렬5’단화3’단분별설계Xho I（CTCGAG）화 Not I（GCGGCCGC）한제성매절위점，장기중조도원핵표체질립 pMD19-T중，득도 pMD19-T-Lys-C，병재필적효모중획득표체。응용 SDS-PAGE 전영급면역반점법검측기표체，RP-GPLC 분석중조뢰안산내태매가이특이성절제뢰안산기단 C 말단적안기산잔기，증명해중조적뢰안산내태매가체대 Lysyl Endopeptidase 응용우이도소적제비공예중。
The amino acid sequence of lysine peptide endopeptidase in peptide was recombined and cDNA 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 cDNA sequence. The sequence was recom-bined to prokaryotic expression plasmid pMD19-T,and pMD19-T-Lys-C was obtained and expression was acquired in pichia pastoris. SDS-PAGE and dot immunobinding assay were used in the determina-tion of expression. RP-GPLC analysis proved that amino acid residue at C end of lysine groups can be excised specifically by lysine peptide endopeptidase recombination. Lysine peptide endopeptidase re-combination can replace Lysyl Endopeptidase in the preparation technology of insulin.