High-Level Expression of Rat Farnesyl:Protein Transferase inEscherichia colias 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 of a variety of cellular proteins. Since FPTase is a large (95-kDa) heterodimeric protein and is inactive unless the α- and β-subunits are coexpressed, large-scale overexpression of active enzyme has been challenging. We report the design of a translationally coupled expression system that will produce FPTase at levels as high as 30 mg/LEscherichia coli.Heterodimeric expression of FPTase was achieved using a translationally coupled operon from the T7 promoter of the pET23a (Novagen) expression plasmid. The β-subunit-coding sequence was placed upstream of the α-subunit coding sequence linked by overlapping β-subunit stop and α-subunit start codons. Additionally, the initial 88 codons of the α-subunit gene were altered, removing rare codons and replacing them with codons used in highly expressed proteins inE. coli.Since previous attempts at recombinantly expressing FPTase inE. colifrom a translationally coupled system have demonstrated that initiation of translation of the α-subunit is poor, we propose that the optimization of the codons at the start of the α-subunit gene leads to the observed high level of recombinant expression.