Simple, rapid, high-purity preparation of recombinant human platelet-derived growth factor-BB

被引：6
作者：
Karumuri, Nagaraju N.
Gangireddy, Srinivasa R.
Narala, Venkata R.
Majee, Sangita S.
Gunwar, Sripad
Reddy, Raju C.
机构：
[1] Univ Michigan, Dept Internal Med, Div Pulm & Crit Care Med, Ann Arbor, MI 48109 USA
[2] Univ Michigan, Dept Biol Chem, Ann Arbor, MI 48109 USA
[3] Virchow Res Inst, Hyderabad 500055, Andhra Pradesh, India
来源：
BIOTECHNOLOGY LETTERS | 2007年 / 29卷 / 09期
关键词：
dimerization; platelet-derived growth factor; protein purification; refolding;
D O I：
10.1007/s10529-007-9411-9
中图分类号：
Q81 [生物工程学（生物技术）]; Q93 [微生物学];
学科分类号：
071005 ; 0836 ; 090102 ; 100705 ;
摘要：
Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) is used to treat full-thickness diabetic ulcers and is being investigated for use in other chronic ulcers, non-healing wounds, and periodontal defects. A simple, novel method for expression and purification of rhPDGF-BB from Escherichia coli is now described. This method produces the dimeric protein in high yield (10-12 mg/g wet cell mass) and with a purity > 95%. rhPDGF-BB was exclusively found in inclusion bodies (IBs) representing approx. 30% of the total cell proteins. The IBs were extracted and the monomer purified by RP-HPLC. The purified rhPDGF-B monomer was then refolded using Tris buffer and subsequently dimerized to produce biologically active rhPDGF-BB. This product was composed of two polypeptide chains, each approx. 12 kDa. The final product exhibited specific activity in a fibroblast proliferation assay indistinguishable from that of the WHO reference standard.
引用
收藏
页码：1333 / 1339
页数：7
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