Acta Scientiarum Naturalium Universitatis Pekinensis

Previous Articles     Next Articles

Construction, Expression, Purification and Characterization of GHRP-scu-PA-32K

JIAO Jianwei, XU Changfa   

  1. National Laboratory of Protein Engineering and Plant Gene Engineering, Peking University, Beijing, 100871
  • Received:1999-03-01 Online:2000-03-20 Published:2000-03-20


焦建伟, 徐长法   

  1. 北京大学蛋白质工程及植物基因工程国家重点实验室,北京,100871

Abstract: GHRP-scu-PA-32K cDNA was obtained by in vitro site-directed-mutagenesis to insert oligonucleotide sequence (GGTCATAGGCCT) encoding tetrapeptide Gly-His-Arg-Pro into scu-PA-32K gene at site just preceding the amino-terminal residue(Leu1). The mutant cDNA was cloned in pCM-β-neo expression vector and co-transformed CHO-DHFR- cells together with pCM-dhfr. The stable expression cell line was selected. Expression level of the line cultured in serum-free medium was 580IU/106 cell/24h. The product expressed was purified by Zn2+-Sepharose affinity chromatography. The apparent molecular weight of purified GHRP-scu-PA-32K was 33kD by SDS-PAGE and mass spectrometry and its specific activity was 150000IU/mg protein, according to fibrin plate determination. The affinity for fibrin of GHRP-scu-PA-32K was 4.5 times higher than that of scu-PA-32K.

Key words: scu-PA, CHO cell, tetrapeptide GHRP, site-directed mutagenesis

摘要: 在scu-PA-32K的cDNA分子基础上经定点突变,在N端紧接Leu1之前引入编码GHRP四肽的寡核苷酸序列(GGTCATAGGCCT),构建了GHRP-scu-PA-32K的突变体cDNA。将它克隆到表达载体pCM-βneo中,与pCMβ-dhfr共转染CHO/DHFR-细胞。筛选到的稳定表达株在无血清培养基的表达量为580IU/(106细胞·24h)。经锌离子螯合亲和柱纯化的产物,SDS-PAGE显示为单一蛋白条带,分子量为33kD,质谱法测定分子量也为33kD。经血纤维蛋白平板法测定比活为150000IU/mg蛋白。对血纤维蛋白的亲和性,突变体为scu-PA-32K的4.5倍。

关键词: 单链尿激酶型纤溶酶原激活剂, GHRP四肽, CHO细胞, 定点突变

CLC Number: