Expression of lipase-solubilized bovine liver microsomal cytochrome b(5) in Escherichia coli as a glutathione S-transferase fusion protein (GST-cyt b(5))

摘要

The gene coding for the lipase-solubilized bovine liver microsomal cytochrome b(5) (cyt b(5)) was expressed in Escherichia coli BL21 cells as a glutathione S-transferase fusion protein (GST-cyt b(5)) using the constructed expression vector pGEX-cyt b(5). The GST-cyt b(5) fusion protein can be matured in vivo as a holoprotein with heme incorporated into cyt b(5) during the fermentation, and the purification procedures were simplified by using a one-step affinity column chromatography with glutathione-agarose gel. The fusion protein was characterized by its spectroscopic and electrochemical properties, the interaction between GST-cyt b(5) and cyt c was also investigated. The results show that GST-cyt b(5) fusion protein shares similar properties and functions to that of isolated cyt b(5). Although cyt b(5) and GST were fused together, the two partners have not made significant structural and functional alterations of their counterparts, the protein-protein interactions between them are apparently very weak. To our knowledge, the present study is the first report to express cyt b(5) as a GST-cyt b(5) fusion protein, which provides a good example for the in vivo maturation of a hemoprotein as a GST fusion protein and sheds new light on the protein-protein interactions within the GST fusion protein. (c) 2005 Elsevier Inc. All rights reserved.