Novel method for expression and purification of human pigment epithelium-derived factor with biological activities in Escherichia coli.
We developed a novel method for the expression and purification of recombinant human PEDF in Escherchia coli, and proved it to be simple, convenient, and cheap to obtain this protein with biological activity intact. Human PEDF gene, amplified by PCR from human retinal cNDA library, was cloned into the prokaryotic expression vector pET-22b(+). The recombinant pET-22b(+)/PEDF was expressed in E. coli strain BL21(DE3). The recombinant protein showed a molecular weight of about 50 kDa and was mainly in the form of inclusion bodies according to SDS-PAGE and Western blot analysis. The insoluble rPEDF was solublized from inclusion bodies by denaturation using 6 M urea, purified by His-tag affinity chromatography, and renatured to natural structure by dialysis in the presence of DTT. The rPEDF could cell-type-specifically inhibit HRCEC proliferation in a dose-dependent manner and induce HRCEC apoptosis.
Wang
Qing-Song
QS
Department of Biochemistry, Zhongshan Medical School, Sun Yat-sen University, Guangzhou, Guangdong Province, P. R. China.
Yang
Xia
X
Yang
Zhong-Han
ZH
Gao
Guo-Quan
GQ
D049109:Cell Proliferation / Q000187:drug effects; D004926:Escherichia coli / Q000378:metabolism; D005136:Eye Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D007218:Industrial Microbiology / Q000379:methods*; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000187:drug effects; D010957:Plasmids / Q000235:genetics; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D015843:Serpins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology; D000099174:Pigment Epithelium-Derived Factor
D005136:Eye Proteins; D009414:Nerve Growth Factors; D011994:Recombinant Proteins; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor