High efficient expression of recombinant human cystatin C in Pichia pastoris
- VernacularTitle:重组人胱蛋白酶抑制剂C在毕氏酵母菌中的高效表达
- Author:
Haixia LI
;
Yulin LI
;
Guobin XU
;
Lihua ZHU
- Publication Type:Journal Article
- Keywords:
Yeast;
Gene expression;
Cystatin C
- From:
Chinese Journal of Laboratory Medicine
2001;0(05):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct human Cystatin C expression vector and make primary protein purification. Methods A 380 bp cDNA fragments encoding Cystatin C were isolated by RT PCR from human placenta tissue and then inserted into pPIC9 vector. Recombinant pPIC9 Cystatin C was transformed into Pichia pastoris strain GS115. Secreted soluble Cystatin C was produced by induction of the AOX1 promoter with methanol. Recombinant Cystatin C was purified by Hitrap SP cation exchange chromatography. Results The expression level of Cystatin C reached 16 mg/L. The stability of Cystatin C is 3 days in expression system. SDS PAGE revealed that Cystatin C was expressed up to 60% of the total soluble protein. After purified by Hitrap SP cation exchange, the recovery rate was 40.5%. Conclusion Cystatin C has a high secretion in Pichia pastoris yeast and could be recognized by polyclonal antibody of native human Cystatin C. After purification,this protein can be used as a standard and antigen to develop immunoassay for human Cystatin C in serum.
