To obtain recombinant human β2-microglobulin (rhβ2M) with properties of good solubility and high purity from E.coli, prokaryotic expression conditions were optimized and protein purification was performed in this study. After testing the effect of different IPTG concentrations, temperatures and induction times on the production of rhβ2M, the optimum expression conditions were determined, i.e. joining IPTG to final concentration being 0.8 mmol/L and inducing time 6 h and at temperature of 25℃. Under the optimum induction conditions, the ratio of soluble rhβ2M to soluble bacterial protein was 63.7%. After purified by Ni Sepharose 6 Fast Flow, the purity of rhβ2M achieved a greater value of 95%. Western blot analysis revealed that rhβ2M possessed the antigen property that specifically interacted with anti-β2M antibody.
In order to construct and express human cardiac troponin C-linker-troponin I(P) fusion protein, detect its activity and prepare lyophilized protein, we searched the CDs of human cTnC and cTnI from GenBank, synthesized cTnC and cTnI(30-110aa) into cloning vector by a short DNA sequence coding for 15 neutral amino acid residues. pColdⅠ-cTnC-linker-TnI(P) was constructed and transformed into E. coli BL21(DE3). Then, cTnC-linker-TnI(P) fusion protein was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). Soluable expression of cTnC-linker-TnI(P) in prokaryotic system was successfully obtained. The fusion protein was purified by Ni2+ Sepharose 6 Fast Flow affinity chromatography with over 95% purity and prepared into lyophilized protein. The activity of purified cTnC-linker-TnI(P) and its lyophilized protein were detected by Wondfo FinecareTM cTnI Test. Lyophilized protein of cTnC-linker-TnI(P) was stable for 10 or more days at 37℃and 4 or more months at 25℃and 4℃. The expression system established in this research is feasible and efficient. Lyophilized protein is stable enough to be provided as biological raw materials for further research.