High level expression in Saccharomyces cerevisiae of an artificial gene encoding a repeated tripeptide aspartyl-phenylyalanyl-lysine.
A chemically synthesized gene, which encodes a 64 or 128 times-repeated tripeptide, aspartyl-phenylalanyl-lysine, has been cloned onto the yeast expression vector pAM82 containing the PHO5 promoter. The artificial gene (LAP gene) contains the untranslated leader sequence of the E. coli lipoprotein gene (lpp) with its transcription terminator sequence. When yeast AH22 cells transformed by recombinant plasmid containing repeated tripeptide gene were derepressed in low phosphate medium, the artificial polypeptides were synthesized to the amounts of about 30% of the total cell protein. SDS-polyacrylamide gel electrophoresis and immunoblot analysis indicated that the artificial polypeptides synthesized in yeast have molecular weights ranging from about 30,000 and 60,000 and have immunoreactivity with the artificial polypeptides expressed in E. coli. The artificial popypeptides in whole cell extract were insoluble and seem to be synthesized as insoluble aggregates. Electron microscopy showed the presence of inclusion bodies in the cell. These polypeptides can be hydrolyzed to tripeptides with trypsin or chymotrypsin. These properties along with the high expression and easy separation may make the artificial polypeptides a potential raw material for the production of an artificial sweetener, Aspartame.
Choi
S Y
SY
Department of Agricultural Chemistry, Korea University, South Korea.
Lee
S Y
SY
Bock
R M
RM
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D005813:Genes, Synthetic; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011506:Proteins / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D004274:DNA, Recombinant; C083313:LAP16 protein, S cerevisiae; C083314:LAP32 protein, recombinant; D011506:Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins