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


CONCLUSIONS
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.
Wang
Li-li
LL
Institute of Modern Separation Sciences, Northwest University, Xi'an 710069, China.
Geng
Xin-du
XD
Han
Hua
H
D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D003001:Cloning, Molecular; D003470:Culture Media; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005784:Gene Amplification; D006801:Humans; D007947:Leukemia, Megakaryoblastic, Acute / Q000473:pathology; D010957:Plasmids; D017510:Protein Folding; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D019089:Stem Cell Factor / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
D003470:Culture Media; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D019089:Stem Cell Factor