Vallejo Luis Felipe, Rinas Ursula
Biochemical Engineering Division, GBF German Research Center for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany.
Biotechnol Bioeng. 2004 Mar 20;85(6):601-9. doi: 10.1002/bit.10906.
The human gene encoding the mature form of bone morphogenetic protein-2 (hBMP-2), a dimeric disulfide-bonded protein of the cystine knot growth factor family, was expressed in recombinant Escherichia coli using a temperature-inducible expression system. The recombinant protein was produced in the form of cytoplasmic inclusion bodies and the effect of different variables on the renaturation of rhBMP-2 was investigated. In particular, variables such as pH, redox conditions, protein concentration, temperature, the presence of different types of aggregation suppressors, and host cell contaminants were studied with respect to their effect on aggregation during refolding and on the final renaturation yield of rhBMP-2. It is shown that the renaturation yield is particularly sensitive to pH, temperature, protein concentration, and the presence of aggregation suppressors. In contrast, little effect of the redox conditions and the ionic strength on the renaturation yield was observed, as equal yields were obtained in a broad range of reduced to oxidized glutathione ratios and concentrations of NaCl, respectively. The aggregation suppressor 2-(cyclohexylamino)ethanesulfonic acid (CHES) proved to be superior with respect to the final renaturation yield, although, in comparison to the more common arginine, it was less efficient in preventing aggregation of rhBMP-2 during refolding. Detergent washing of inclusion bodies was sufficient, as further purification of rhBMP-2 prior to refolding was without effect on the final renaturation yield. An increase in the concentration of renatured rhBMP-2 was achieved by a pulsed refolding procedure by which up to a total amount of 2.1 mg mL(-1) rhBMP-2 could be transferred in seven pulses into the renaturation buffer with an overall refolding yield of 38%, corresponding to 0.8 mg mL(-1) renatured dimeric rhBMP-2. Furthermore, a simplified purification procedure is presented that also includes freeze-drying for long-term storage of biologically active rhBMP-2. Finally, it is shown that the appearance of rhBMP-2 variants could be avoided by using a host strain overexpressing rare codon tRNAs.
编码骨形态发生蛋白2成熟形式（hBMP - 2）的人类基因，一种胱氨酸结生长因子家族的二聚体二硫键结合蛋白，使用温度诱导表达系统在重组大肠杆菌中表达。重组蛋白以细胞质包涵体的形式产生，并研究了不同变量对rhBMP - 2复性的影响。特别地，研究了诸如pH、氧化还原条件、蛋白质浓度、温度、不同类型聚集抑制剂的存在以及宿主细胞污染物等变量对复性过程中聚集的影响以及对rhBMP - 2最终复性产率的影响。结果表明，复性产率对pH、温度、蛋白质浓度和聚集抑制剂的存在特别敏感。相比之下，未观察到氧化还原条件和离子强度对复性产率有显著影响，因为在广泛的还原型谷胱甘肽与氧化型谷胱甘肽比例范围以及NaCl浓度下分别获得了相同的产率。聚集抑制剂2 -（环己基氨基）乙磺酸（CHES）在最终复性产率方面表现出色，尽管与更常用的精氨酸相比，它在防止rhBMP - 2复性过程中聚集方面效率较低。包涵体的去污剂洗涤就足够了，因为在复性前对rhBMP - 2进行进一步纯化对最终复性产率没有影响。通过脉冲复性程序提高了复性rhBMP - 2的浓度，通过该程序，高达总量2.1 mg mL⁻¹的rhBMP - 2可以分七次脉冲转移到复性缓冲液中，总体复性产率为38%，相当于0.8 mg mL⁻¹复性的二聚体rhBMP - 2。此外，还提出了一种简化的纯化程序，该程序还包括冻干以长期储存具有生物活性的rhBMP - 2。最后，结果表明，通过使用过表达稀有密码子tRNA的宿主菌株可以避免rhBMP - 2变体的出现。
