N-terminal deletions and His-tag fusions dramatically affect expression of cytochrome p450 2C2 in bacteria.
The expression of mutants with deletions in the N-terminal signal-anchor sequence of cytochrome P450 2C2 and His-tag fusions was examined in Escherichia coli to determine the influence of N-terminal sequences on expression of the protein. Two mutants predicted to be translocated across the membrane inhibited bacterial growth. In other mutants, deletion of the N-terminal transmembrane domain (residues 2-20) reduced expression of functional P450 by about 75% and further deletion of the following linker sequence (residues 21-27) resulted in a modest further decrease. Expression of the mutant with residues 2-27 deleted contrasts with the lack of expression of functional protein if only the linker was deleted, which suggests that the linker sequence is critical for expression only if the protein is inserted into the membrane by the transmembrane domain. Fusion proteins of green fluorescent protein with full-length P450 2C2 and 2C2(Delta2-20) were predominantly membrane-associated in vivo as determined by fluorescence microscopy. Subcellular fractionation of bacteria expressing these proteins and extraction of the proteins from the membrane by high salt or alkaline buffer demonstrated that P450 2C2 was an integral membrane protein while 2C2(Delta2-20) was a peripheral membrane protein that associated with the membrane mainly by hydrophobic interactions. Residues 1-27 of P450 2C2 fused to green fluorescent protein resulted in a redistribution of fluorescence from cytosol to membrane, which, with the deletion studies, indicates that the P450 signal-anchor is both necessary and sufficient for normal membrane targeting and is the sole transmembrane domain of cytochrome P450 2C2 in bacteria. Addition of a His-tag at the N-terminus completely restored wild-type expression levels to the 2C2(Delta2-20) mutants in bacteria. In insect cells, functional 2C2(Delta2-20) was not expressed but an N-terminal His-tag also restored full expression. The increase in expression may be related to decreased association with the membrane mediated by the His-tag.
Doray
B
B
Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Chen
C D
CD
Kemper
B
B
D000595:Amino Acid Sequence; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007313:Insecta; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003577:Cytochrome P-450 Enzyme System; C084930:cytochrome P-450 2C2