N-terminal deletions and His-tag fusions dramatically affect expression ofcytochrome P4502C2 in bacteria

The expression of mutants with deletions in the N-terminal signal-anchor se quence of cytochrome P450 2C2 and His-tag fusions was examined in Escherich ia 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 tr ansmembrane 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 sequ ence is critical for expression only if the protein is inserted into the me mbrane by the transmembrane domain. Fusion proteins of green fluorescent pr otein with full-length P450 2C2 and 2C2(Delta2-20) were predominantly membr ane-associated in vivo as determined by fluorescence microscopy. Subcellula r fractionation of bacteria expressing these proteins and extraction of the proteins from the membrane by high salt or alkaline buffer demonstrated th at P450 2C2 was an integral membrane protein while 2C2(A2-20) was a periphe ral membrane protein that associated with the membrane mainly by hydrophobi c interactions. Residues 1-27 of P450 2C2 fused to green fluorescent protei n resulted in a redistribution of fluorescence from cytosol to membrane, wh ich, with the deletion studies, indicates that the P450 signal-anchor is bo th necessary and sufficient for normal membrane targeting and is the sole t ransmembrane domain of cytochrome P450 2C2 in bacteria. Addition of a His-t ag 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 express ion. The increase in expression may be related to decreased association wit h the membrane mediated by the His-tag. (C) 2001 Academic Press.