title: A purification method for tag-free human cystatin C recombinant protein expressed in Escherichia coli.
abstract: To obtain recombinant cystatin C (CysC) protein, which can be used in immunological diagnostic kits, we focused on the preparation of tag-free CysC. The 6 × His-TF-CysC fusion protein was found to overexpress in soluble form in cells of BL21-Gold (DE3)/pCold TF-CysC, which had been induced with isopropyl-D-1-thiogalactopyranoside. Subsequently, we established a protein purification method for tag-free CysC using immobilized metal-affinity chromatography and size-exclusion chromatography. In this method, glutathione-S-transferase-human rhinovirus 3C proteases were used to remove the protein tags. High homogeneity of the purified CysC was determined by SDS-PAGE, while the purity of the tag-free CysC was ascertained to be above 95%. With a yield of 25 mg/L from bacterial culture, the biological activity of the tag-free CysC was evaluated as inhibitors like natural CysC. The performance of this purification method was successfully evaluated in the preparation of other low molecular weight heterologous proteins in Escherichia coli.
authors.affiliation: a Department of Laboratory Medicine , The First Affiliated Hospital of Chongqing Medical University , Chongqing , China.
authors.forename: Te
authors.initials: T
authors.lastname: Chen
authors.affiliation: b Laboratory of Medical Diagnostics (Ministry of Education of China) , Department of Key Laboratory Medicine, Chongqing Medical University , Chongqing , China.
authors.forename: Wenchun
authors.initials: W
authors.lastname: Xu
mesh_terms: D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D055316:Cystatin C / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
chemical_list: D055316:Cystatin C; D011994:Recombinant Proteins
keywords: Cystatin C; in vitro diagnosis; prokaryotic expression; purification method