title: [Expression of recombinant human BMP-6 in Escherichia coli and its purification and bioassay in vitro].
abstract: To purify the recombinant human BMP-6 protein and to establish its in vitro bioassay method. The cDNA encoding the mature peptide of hBMP-6 protein was amplified by reverse transcription-polymerase chain reaction (RT-PCR), using human placental mRNA as template, and subcloned into the high-expression vector pET-15b under the control of T7 lac promoter. The resulting construct, pET-BMP6, was then transformed into an Escherichia coli strain BL21 (DE3) for the production of recombinant hBMP-6 protein (rhBMP-6). After 4 hours of induction by isopropyl-beta-D-thiogalactoside (IPTG), rhBMP-6 (approximately 15kD) was expressed and formed inclusion bodies, contributing up to 10% of the total bacterial protein. The inclusion bodies were isolated and redissolved in 8mol/L urea, and the denatured rhBMP-6 was purified to 95% purity by CM-Cellulose 32 ion exchange chromatography (IEC). The osteoinductivity of rhBMP-6 was measured by the expression of some of the osteoblast differentiation marker genes in rhBMP-6-treated C3H10T1/2 cells as reflected by determinations of alkaline phosphatase (ALPase) activity and semi-quantitative RT-PCR. At the end of the purification process, about 80% of rhBMP-6 formed disulphide-linked homodimers after refolding during renaturation. The apparent size of the protein was 30kD on non-reducing SDS-PAGE, similar to that of the native form of hBMP-6. The enzyme assays showed that the ALPase activity was increased in a dose-dependent manner with the treatment of rhBMP-6. After the addition of 300ng/mL of rhBMP-6, the ALPase activity of C3H10T1/2 cells increased significantly. The activity of rhBMP-6 used was comparable to about 70% of that of the standard hBMP-6 derived from eukaryotic cells. RNA extraction data also showed rhBMP-6 stimulated expression of osteoblast marker genes, including type I collagen, osterix, and osteocalcin in a time-dependent manner. After 5 days of treatment, their level of expression was increased to 3 times that of controls. Bone morphogenetic protein (BMP)-6, a member of the 60A subgroup of the bone morphogenetic protein (BMPs) family, plays a pivotal role in bone formation. Previous evidence showed that BMP-6 is selectively up-regulated by estrogen, suggesting its potential role in the treatment of osteoporotic fractures, especially for menopausal osteoporosis. Our present study demonstrates that the recombinant hBMP-6 produced in Escherichia coli is able to induce pre-osteoblastic cells to differentiate into osteoblasts in vitro, and analysis of mRNA expression of type I collagen, osterix, and osteocalcin can be also a method for measuring the osteoinductivity of BMP. This provides the basis for further studies on ectopic bone formation in the body and for the development of auxiliary drugs for the treatment of osteoporotic fractures.
authors.affiliation: Medical Molecular Genetics Lab, Medical College of Nankai University, Tianjin 300071, China.
authors.forename: Ju-Hua
authors.initials: JH
authors.lastname: Yang
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.forename: Shuang
authors.initials: S
authors.lastname: Yang
authors.forename: Shuang-Qing
authors.initials: SQ
authors.lastname: Wu
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Tian-Hui
authors.initials: TH
authors.lastname: Zhu
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D055418:Bone Morphogenetic Protein 6 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D051379:Mice; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C525816:BMP6 protein, human; D055418:Bone Morphogenetic Protein 6; D007544:Isopropyl Thiogalactoside; D000469:Alkaline Phosphatase