Expression and purification of active recombinant human bone morphogenetic 7-2 dimer fusion protein.
Bone morphogenetic proteins (BMPs) have been applied in bone regeneration therapy due to their significant osteogenic activity, however, the complicated processing and high cost in producing recombinant BMP have limited their use in the clinic. In this study, we have developed a simple method to prepare recombinant human BMP7-BMP2 fusion protein with a flexible peptide linker (rhBMP7-2). The rhBMP7-2 protein is expressed efficiently in Escherichia coli, and the denatured protein purified by anion exchange chromatography then refolded by dialysis. The yield was about 6.8 mg per gram of wet cell weight. The bioactivity of re-folded rhBMP7-2 was measured by alkaline phosphatase assay and alizarin red staining using both C2C12 and MC3T3-E1 cells, and also using the rat subcutaneous ectopic bone formation model. High level osteogenic activity was found in all the assays tested demonstrating the production of corrected folded and active rhBMP7-2 protein.
School of Biotechnology, Tianjin University of Science and Technology, Key Lab of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin 300457, China.
Jianli
J
Dang
School of Biotechnology, Tianjin University of Science and Technology, Key Lab of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin 300457, China.
Lei
L
Jing
School of Biotechnology, Tianjin University of Science and Technology, Key Lab of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin 300457, China.
Weiwei
W
Shi
School of Biotechnology, Tianjin University of Science and Technology, Key Lab of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin 300457, China.
Ping
P
Qin
School of Biotechnology, Tianjin University of Science and Technology, Key Lab of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin 300457, China.
Yuyin
Y
Li
School of Biotechnology, Tianjin University of Science and Technology, Key Lab of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin 300457, China. Electronic address: diaoaipo@tust.edu.cn.
Aipo
A
Diao
D000595:Amino Acid Sequence; D000818:Animals; D055419:Bone Morphogenetic Protein 7 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D005260:Female; D002479:Inclusion Bodies; D051379:Mice; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000187:drug effects; D010012:Osteogenesis / Q000187:drug effects*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*
C525819:BMP7 protein, human; D055419:Bone Morphogenetic Protein 7; D011993:Recombinant Fusion Proteins
Dialysis; Inclusion body; Osteogenesis; Protein refolding; rhBMP7-2