High-level expression of rat farnesyl : protein transferase in Escherichiacoli as a translationally coupled heterodimer

Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid group from farnesyl diphosphate to the CaaX cysteine o f a variety of cellular proteins. Since FPTase is a large (95-kDa) heterodi meric protein and is inactive unless the alpha- and beta-subunits are coexp ressed, large-scale overexpression of active enzyme has been challenging. W e report the design of a translationally coupled expression system that wil l produce FPTase at levels as high as 30 mg/L Escherichia coli. Heterodimer ic expression of FPTase was achieved using a translationally coupled operon from the T7 promoter of the pET23a (Novagen) expression plasmid. The beta- subunit-coding sequence was placed upstream of the alpha-subunit coding seq uence linked by overlapping beta-subunit stop and alpha-subunit start codon s, Additionally, the initial 88 codons of the alpha-subunit gene were alter ed, removing rare codons and replacing them with codons used in highly expr essed proteins in E. coli. Since previous attempts at recombinantly express ing FPTase in E. coli from a translationally coupled system have demonstrat ed that initiation of translation of the alpha-subunit is poor, we propose that the optimization of the codons at the start of the alpha-subunit gene leads to the observed high level of recombinant expression. (C) 1998 Academ ic Press.