Stability analysis of CHO cell line expressing heterologous rhBMP2 / 生物工程学报

Bone morphogenetic protein 2 (BMP2), which belongs to the transforming growth factor-beta (TGF-beta) superfamily, is a multifunctional molecule with distinct abilities to induce bone formation. BMP2 has been identified to have eminent pharmaceutical importance for clinical application. We previously constructed stable cell line in Chinese hamster ovary cells (CHO) that highly expressed recombinant human BMP2 (rhBMP2). For large-scale production of the recombinant protein used in clinical application, it is critical to have both high expression and stability of the protein. In the present study, the stability of the cell line (rCHO(hBMP2)-C8) with the highest expression, as well as the stability of rhBMP2 protein were investigated systematically. We cultured the rCHO (hBMP2)-C8 cell line in the presence or absence of MTX for two months, the cell growth and rhBMP2 production characteristics were examined during the culture; we found the duration that the rCHO(hBMP2)-C8 cell line could secret rhBMP2 continually into the serum-free medium. Moreover, we detected the temperature sensitivity of rhBMP2 in culture medium. This study will contribute to our understanding for further producing rhBMP2 by large-scale culture technology.

Effect of different tags on pulldown assays implemented by LMO2 fusion protein / 生物工程学报

Pulldown assay is an in vitro method for studies of protein-protein interactions, in which tagged proteins are usually expressed as the bait to enrich other proteins that could bind to them. In this technology, the GST tag is broadest used for its modest size and hydrophilic property. In most cases, the GST tag could increase the hydrophility of the fusion protein and help to avoid the formation of inclusion bodies. However, in the other few cases, the target protein may be strongly hydrophobic or have complicated structures that were hard to fold and assemble in correct conformations without champerons, and even the existence of GST tag could not make them soluble. These proteins were always expressed as inclusion bodies and had no functions. LMO2 was a small molecular weight and insoluble protein, in this study, GST system and MBP system were used to express GST-LMO2 and MBP-LMO2 fusion proteins, respectively. We found that GST-LMO2 fusion protein was expressed as inclusion bodies whereas MBP-LMO2 fusion protein was expressed in soluble form. Moreover, the production rate of MBP-LMO2 was also much higher than GST-LMO2. Then MBP-LMO2 fusion proteins and renatured GST-LMO2 fusion proteins were used as bait in pulldown assay to study the interaction between LMO2 and endogenous GATA1 in K562 cells. Western blot analyses showed that both of these proteins could bind to endogenous GATA1 in K562 cells, but recovered GATA1 protein by MBP-LMO2 fusion protein was much more than GST-LMO2 fusion protein. These results suggest that using of MBP system is a helpful attempt in the case of studying small molecular weight, strong hydrophobic proteins.