Expression of lipase-solubilized bovine liver microsomal cytochrome b5 in Escherichia coli as a glutathione S-transferase fusion protein (GST-cyt b5).
The gene coding for the lipase-solubilized bovine liver microsomal cytochrome b5 (cyt b5) was expressed in Escherichia coli BL21 cells as a glutathione S-transferase fusion protein (GST-cyt b5) using the constructed expression vector pGEX-cyt b). The GST-cyt b5 fusion protein can be matured in vivo as a holoprotein with heme incorporated into cyt b5 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 b5 and cyt c was also investigated. The results show that GST-cyt b5 fusion protein shares similar properties and functions to that of isolated cyt b5. Although cyt b5 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 b5 as a GST-cyt b5 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.
Lin
Ying-Wu
YW
Chemical Biology Lab, Department of Chemistry, Fudan University, Shanghai 200433, PR China.
Zhao
Dong-Xin
DX
Wang
Zhong-Hua
ZH
Yu
Wen-Hao
WH
Huang
Zhong-Xian
ZX
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D015786:Cytochromes b5 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D008049:Lipase / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D045304:Cytochromes c; D015786:Cytochromes b5; D005982:Glutathione Transferase; D008049:Lipase