Heterologous expression of cytochrome P-450 in Escherichia coli.
While the bacterial expression system has proved to work well for the expression of functional forms both of microsomal and of mitochondrial P-450, we understand the requirements for expression in bacteria far less clearly than those for expression in eukaryotic cells. Nevertheless, the ability to generate large quantities of P-450 (Table 1) in an inexpensive culture system, the ease of site-directed mutagenesis in bacteria and the ability to purify easily relatively large quantities of P-450s, including human enzymes that are not normally available, make this a particularly useful expression system for P-450 structure/function analysis. Furthermore, the background of P-450 level in E. coli is extremely low. It appears that the minor sequence alterations that have been made at the N-terminus to achieve high expression do not alter the enzymic properties of the recombinant P-450s, and it will not be surprising if vector systems are developed where such changes are not necessary. One concern with bacterial expression of P-450 is whether this system will prove of general use. Preliminary data suggests that certain mutant forms of human P-450c17 that are known to be functional in COS cells are not functional in bacteria, suggesting further that the folding pathways are not the same in bacterial and in eukaryotic cells. This unpredictability of the bacterial system is unsettling and, only as more laboratories adopt this system because of its value for high-level P-450 expression, will we learn more details of the requirements and limitations of this system.(ABSTRACT TRUNCATED AT 250 WORDS)
Waterman
M R
MR
Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, Tennessee 37232-0146.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D014162:Transfection
D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System