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Published on: March 10, 2012
Efficient expression, purification and characterization of native human cystatin C in Escherichia coli periplasm
Yongjun Zhou1, Yan Zhou2, Jun Li1
1State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Medical School, West China Hospital, Sichuan University, Chengdu, PR China.
Abstract:
Human cystatin C (HCC), encoded by cystatin 3 gene, is a 13.3kDa endogenous cysteine proteinase inhibitor and an important biomarker of renal function. However, expressing recombinant cystatin C is difficult because of low yield and inclusion bodies in Escherichia coli (E. coli). In this study, we cloned HCC gene into pET-22b vector containing PelB leader signal sequence, which could direct the protein to the bacterial periplasm. Large amounts of soluble HCC could be efficiently expressed in the bacterial periplasm at 16°C with 0.1mM IPTG induction. The recombinant HCC was isolated in high purity by cation exchange chromatography and gel filtration chromatography. Furthermore, the HCC was characterized by circular dichroism (CD) and dynamic light scattering (DLS), and displayed biological activity against papain. Here, we provide a method to produce large amounts of soluble mature HCC in E. coli.
