Objective To investigate prokaryotic recombinant expression and purification of human cystatin C. Methods The cDNA sequence of human cystatin C was obtained by RT-PCR amplification , and then constructed into expression vector pET-32a (+). To explore the best expression condition, the pET-32a(+) vectors were transformed into expression host strain BL21(DE3), and the expression condition was optimized. The human cystatin C recombinant protein was purified and obtained by Ni-chelating affinity chromatography and DEAE-sepharose FF ion-exchange chromatography. Results The cDNA sequence obtained by RT-PCR amplification was confirmed as expected by sequencing. By optimization, the expression level of Cys-C has reached to 160 mg/L, accounting for about 20% of total soluble proteins. Followed by a two-step purification, the recombinant Cys-C product was obtained with a purity of 95%. Conclusions The Cys-C prokaryotic expression and protein purification system was constructed successfully, laying the foundation for the following preparation of polyclonal antibodies and the development of in-vitro diagnostic kits.
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