High-Level Expression of Rat Farnesyl:Protein Transferase in Escherichia coli as a Translationally Coupled Heterodimer

Author： Zimmerman K.K. Scholten J.D. Huang C-C. Fierke C.A. Hupe D.J.

ISSN： 1046-5928

Source： Protein Expression and Purification, Vol.14, Iss.3, 1998-12, pp. : 395-402

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Abstract

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 - and &bgr;-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/L Escherichia coli. Heterodimeric expression of FPTase was achieved using a translationally coupled operon from the T7 promoter of the pET23a (Novagen) expression plasmid. The &bgr;-subunit-coding sequence was placed upstream of the &agr;-subunit coding sequence linked by overlapping &bgr;-subunit stop and &agr;-subunit start codons. Additionally, the initial 88 codons of the &agr;-subunit gene were altered, removing rare codons and replacing them with codons used in highly expressed proteins in E. coli. Since previous attempts at recombinantly expressing FPTase in E. coli from a translationally coupled system have demonstrated that initiation of translation of the -subunit is poor, we propose that the optimization of the codons at the start of the -subunit gene leads to the observed high level of recombinant expression.