MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies

Microbial Cell Factories

Research

Subin Kim¹  Yun-Hui Jeon¹  Phuong Hong Nguyen¹  So-Mi Yoon¹  Senyon Choe²  MarioMeng-Chiang Kuo² 

[1] Protein Engineering Laboratory, joint Center for Biosciences, Songdo Smart Valley, 214 Sondgo-dong, 406-840, Yeonsu-gu, Incheon, Korea;Protein Engineering Laboratory, joint Center for Biosciences, Songdo Smart Valley, 214 Sondgo-dong, 406-840, Yeonsu-gu, Incheon, Korea;Drug Discovery Collaboratory, Carlsbad Center for Translational Medicine, 92008, Carlsbad, CA, USA;

关键词: TGF-beta;    BMP-9;    MB109;    Refolding;    Rapid dilution;    E. coli;    Recombinant cytokine;   

DOI  :  10.1186/1475-2859-13-29

 received in 2013-12-05, accepted in 2014-02-14,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundThe development of chemical refolding of transforming growth factor-beta (TGF-β) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-β superfamily ligands could not be refolded readily by the same methods.ResultsHere, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 – Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9.ConclusionMB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of aggregation suppressors and the concentrations of protein, salt and detegent. These results add to the current understanding of producing recombinant TGF-β superfamily ligands in the microbial E. coli system. An application of the technique to produce a large number of synthetic TGF-β chimeras for activity screen is also discussed.

【 授权许可】

CC BY   
© Kuo et al.; licensee BioMed Central Ltd. 2014