High-level expression of rat farnesyl:protein transferase in Escherichia coli 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 of a variety of cellular proteins. Since FPTase is a large (95-kDa) heterodimeric protein and is inactive unless the alpha- and beta-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 beta-subunit-coding sequence was placed upstream of the alpha-subunit coding sequence linked by overlapping beta-subunit stop and alpha-subunit start codons. Additionally, the initial 88 codons of the alpha-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 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.
Zimmerman
K K
KK
Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan, 48105, USA.
Scholten
J D
JD
Huang
C C
CC
Fierke
C A
CA
Hupe
D J
DJ
D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D003062:Codon / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D051231:Farnesyltranstransferase; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D018411:Spodoptera / Q000166:cytology
D003062:Codon; D011993:Recombinant Fusion Proteins; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase