[Cloning, expression, renaturation and purification of soluble hSCF].

AIM

To construct an expression vector for soluble recombinant human stem cell factor (rhSCF) gene, and optimize culture conditions for high-level expression of rhSCF in E.coli.

METHODS

hSCF cDNA was amplified by PCR with total cDNA from human lymph node as a template, followed by cloning into pMD18-T vector. The 5' terminal of the hSCF cDNA was modified by degenerative PCR to obtain high-level expression in E.coli. The biological activity of the refolded rhSCF, purified with high performance hydrophobic interaction chromatography, was examined by MTT colorimetry.

RESULTS

hSCF cDNA was amplified and cloned, and was inserted into an E.coli expression vector pBV220. The expressed rhSCF accounted for about 20% of total bacterial proteins and reached 40% of total bacterial proteins under optimal culture conditions. The expressed rhSCF appeared in bacterial lysates in the form inclusion body. The rhSCF with biological activity was obtained after solubilization of the inclusion body with 8 mol/L urea or 7 mol/L guanidine chloride, followed by preliminary refolding and purification.

CONCLUSION

hSCF was cloned and expressed in E.coli successfully. The E.coli strain expressing rhSCF can be used to produce rhSCF with biological activity on large-scale production.