Simple, rapid, high-purity preparation of recombinant human platelet-derived growth factor-BB.
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.
Karumuri
Nagaraju N
NN
Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Gangireddy
Srinivasa R
SR
Narala
Venkata R
VR
Majee
Sangita S
SS
Gunwar
Sripad
S
Reddy
Raju C
RC
D000077214:Becaplermin; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods; D006801:Humans; D010982:Platelet-Derived Growth Factor / Q000008:administration & dosage / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins / Q000378:metabolism
D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D011994:Recombinant Proteins; D000077214:Becaplermin