中国生物工程杂志

Objective: Clone, express and purify human recombinant calreticulin (CRT). Methods: Human CRT cDNA was amplified by RT-PCR from total RNA of human lung cancer cell line A549 cells. Then, PCR product was subcloned into prokaryotic expression vector pET-15b. After sequencing, this recombinant plasmid was transformed into E. coli. Rossetta. Recombinant CRT was expressed in host cells by IPTG induction. Resulted protein was purified by Ni-NTA resin under denature condition and dialyzed to recover its native structure. SDS-PAGE and Western blot method were used to identify the expression and purification of reconbinant CRT. Results: Human CRT cDNA was cloned from total RNA of A549 cells. CRT prokaryotic expression vector pET-15b-crt was constructed. Reconbinant CRT was induced to express in E. coli and purified by Ni-NTA affinity chromatograph. Conclusion: A method for prokaryotic expression and purification of human recombinant CRT was successfully established. This method laid a foundation for the succedent CRT research.