ABSTRACT
Objective:To construct the Escherichia coli (E. coli)expression system for preparation of the bone morphogenetic protein-2 (BMP2)with collagen-binding domain (CBD),and to study the methods and conditions for expression, purification and renaturation of CBD-BMP2.Methods:CBD sequence was cloned into the N-terminal of BMP2 sequence, the recombinant vector pet21b/CBD-BMP2 was constructed and transformed into E.coli BL21.The expression of recombinant protein was induced using isopropylβ-D-thiogalactopyranoside (IPTG) at 37 ℃.Ni-NTA chelate chromatography was used to purify CBD-BMP-2.Denaturing CBD-BMP2 was refolded by dilution method using ultrapure water.The refolding CBD-BMP2 was filtered through a 0.22μm microfiltration membrane for degermation.The recovery rate was calculated by the ratio of the protein concentration before and after degermation. The expression, purification, and renaturation of recombinant protein were detected by SDS-PAGE method.The concentration of CBD-BMP2 was detected by BCA assay.Results:The recombinant vector pet21b/CBD-BMP2 was successfully transformed into E.coli BL21,and the recombinant protein was expressed as inclusion bodies in E.coli.The SDS-PAGE results showed denaturing protein was dissolved in supernatant of lysis buffer with 8 mol·L-1 urea and the purified recombinant protein existed in elution buffer B with relative molecular mass about 14 000.Two bands (14 000 and 28 000)were seen in the SDS-PAGE picture,which indicated that the monomer was successfully refolded into dimer by dilution method.The concentrations of recombinant protein before and after degermation were 110 and 80 mg · L-1 , respectively, and the recovery rate was about 73%. Conclusion:The recombinant vector pet21b/CBD-BMP2 is transformed into E.coli BL21 successfully,and the recombinant CBD-BMP2 is expressed and refolded efficiently. The methods of prokaryotic expression system for preparing recombinant CBD-BMP2 protein are established.
ABSTRACT
BACKGROUND: Several studies have confirmed that activation of intervertebral disc enzymes is closely related to matrix degradation. Matrix metalloproteinase and tissue inhibitor of metalloproteinase have been shown to exert important roles in the process of extracellular matrix degeneration in intervertebrel disk. Besides these two enzyme systems, whether other proteeses that exhibit degrading effects on extracellular matrix are involved in the intervertebral disk degeneration remains poorly understood.OBJECTIVE: To detect the cathepsin K expression in normal and degenerated human intervertebrel disc cells and investigate the correlation between cathepsin K and intervertebral disc degeneration.METHODS: Cathepsin K expression was detected in intervertebral disc tissue from 30 patients with lumbar intervertebral disc protrusion using immunohistochemistry SP method and ELISA. At the same time, the intervertebral disc tissue from 15 healthy adult cadavers and/or spine fracture patients was taken as control, Cathepsin K protein expression in normal and degenerated human intervertebral disc tissues were compared.RESULTS AND CONCLUSION: Cathepsin K expression was observed in normal and degenerated intervertebral disc tissues.The expression level was significantly higher in degenerated tissue than normal tissue (P ＜ 0.05). These findings demonstrate that Cathepsin K possibly participates in the intervertebral disc degeneration.
ABSTRACT
Objective: To investigate the applied feasibility of scaffold with modified PLA (Polymer of lactic acid) in tissue engineering. Methods:First, we adopted salting-in method to prepare porous foam scaffold. Then, we reconstructed tissue engineering skin by epidermal cells and fibroblasts combined with modified PLA. On the 14th day of cell culturing in vitro, we was a control. Results:The arfificial skin is composed of epidermis and dermis and similar to natural skin in appearance. The skin consists of fibroblasts and keratinocytes, which are in various proliferation and differentiation stages. Fibroblasts and keratinocytes distribute on the surface of polymer of lactic acid (PLA) and the number of fibroblast and keratinocyte increase. Conclusion:Dialdehyde starches (DAS) not only improve the function of PLA but also have good effects on cells. Moreover, it does not affect the growth and the metabolism of the cells. So it is feasible to use modified scaffold to construct tissue engineering skin.