Construction of the complete rat fatty acid synthase cDNA and its expression in Saccharomyces cerevisiae.
The 272 647-dalton polypeptide of fatty acid synthase (FAS) from Rattus norvegicus has been expressed in a proteinase-deficient strain of Saccharomyces cerevisiae. The seven overlapping cDNA clones for rat FAS spanning the entire coding region were the starting material for this undertaking. In a series of cloning steps an expression plasmid was constructed in which the cDNA was placed under the control of the yeast ADH1 promoter. Northern blotting of total RNA isolated from yeast transformed with this expression plasmid demonstrated a high rate of transcription of the 7.4-kb cDNA. However, a successful translation required further manipulation of the sequence immediately upstream of the rat FAS translational start codon. This was obtained when the 86 bp of the rat FAS cDNA immediately 5' to the start codon were replaced by a nonamer corresponding to the immediate 5'-vicinity of the translational start codon of the yeast ADH1 gene. Nevertheless, the translation product could be detected only by Western blotting. The FAS proteins of S. cerevisiae and rat are not functionally interchangeable. Using the purification protocol of rat FAS the heterologously expressed FAS could be enriched by at least one order of magnitude.
Kupfer
R
R
Institut für Biochemie, Universität Erlangen-Nürnberg, Staudtstrasse. 5, D-91058 Erlangen, Germany.
Beiche
F
F
Schweizer
M
M
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D064429:Fatty Acid Synthases / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D018076:DNA, Complementary; D011994:Recombinant Proteins; D064429:Fatty Acid Synthases