Title
Efficient expression, purification and characterization of native human cystatin C in Escherichia coli periplasm
Author
Yongjun Zhou, Yan Zhou, Jun Li, Jian Chen, Yuqin Yao, Lin Yu, Desheng Peng, Mingrong Wang, Dan Su, Yong He, Lantu Gou
Year
2015
Journal
Protein Expression and Purification
Abstract
Human cystatin C (HCC), encoded by cystatin 3 gene, is a 13.3 kDa 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 inEscherichia 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 0.1 mM 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.
Instrument
J-1500
Keywords
Circular dichroism, Secondary structure, Biochemistry