2.2. Construction and identification of recombinant human-like collagen
The gene encoding RHC was cloned into the pET-3c expression vector to generate a recombinant plasmid named pET3c-hlcollagen, which was transformed into E. coliBL21(DE3 ). After screening for ampicillin resistance and induction by IPTG, the best expression condition was selected. Lager scale production of RHC was performed using a 50L fermenter. And RHC protein was purified using affinity chromatography on a NI Sepharose 6 Fast Flow column combined with gel filtration Sephadex G-25. PCR, western blot, and gel electrophoresis were used for identification of RHC.