Downloadable ContentDownload PDF
Purification and Characterization of the Enzyme BaiK
The liver generates primary bile acids for use in the intestinal tract. Once released in the tract, intestinal bacterium convert these primary bile acids to secondary bile acids, which have been correlated to a number of intestinal diseases such as cancers in the GI tract. This conversion occurs via a metabolic pathway encoded by the bile acid induced (bai) operon. One gene, baiK, encodes a putative CoA transferase; however, this gene product has not been characterized. To investigate the role of BaiK, further in vitro characterization is needed, requiring a significant amount of purified protein. To determine expression conditions that maximize the production of soluble BaiK, different E. coli cell lines, temperatures, and induction times were investigated using SDS-PAGE analysis of whole-cell and soluble protein samples. The next step, BaiK purification, used immobilized metal affinity chromatography and size exclusion chromatography to obtain the purified BaiK protein. Using the purified protein, initial crystallization trials were conducted to identify conditions that could lead to crystal formation. Future plans include optimization of any crystallization leads and structure determination, which will allow for function and mechanism of BaiK to be better understood.