Expression And Analysis Of Sls-like Gene In Escherichia

Secologanin synthase (SLS) is one of key rate-limiting enzymes during methabolic synthesis pathway of Camptothecin (CPT). The gene sls-like, which is similar to the gene sls, has been isolated in the prophase from our laboratory. The purpose of this work is to express the SLS protein, which is coded by sls-like gene, in host E.coli BL21(DE21) after inserting this gene into E.coli DH5α. Expressed protein can be isolated, purified and then analysed by HPLC-MS.Our results showed that we have successfully obtained recombinant plasmid pET-28a-sls-like and sls-like gene was expressed in E.coli BL21(DE21). Over-expression proteins was induced by T7 promoter regulation and inducer IPTG with 0.6 mmol/L at 28℃for 3h of culture. However, it was inclusion body proteins. Purification with (His)6-tagged proteins (IMAC) using Chelating SepharoseTM Fast Flow was carried out. Finally, purified proteins, which were confirmed by using high-performance liquid chromatography and mass spectrometry, were proved to be target proteins. In addition, data indicated that the target protein has a sequence similarity of 62.8% with SLS. This result is consistant with that predicted in the previous work from our laboatory.