Back to Search
Start Over
High level expression in Saccharomyces cerevisiae of an artificial gene encoding a repeated tripeptide aspartyl-phenylyalanyl-lysine
- Source :
- Journal of biotechnology. 30(2)
- Publication Year :
- 1993
-
Abstract
- 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 immunore-activity 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 hydrolized 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.
- Subjects :
- Saccharomyces cerevisiae Proteins
Saccharomyces cerevisiae
Immunoblotting
Molecular Sequence Data
DNA, Recombinant
Bioengineering
Tripeptide
Biology
Molecular cloning
Applied Microbiology and Biotechnology
Plasmid
Gene expression
Genes, Synthetic
Amino Acid Sequence
Cloning, Molecular
Repetitive Sequences, Nucleic Acid
Expression vector
Chymotrypsin
Base Sequence
Proteins
General Medicine
biology.organism_classification
Molecular biology
Yeast
Recombinant Proteins
Kinetics
Biochemistry
biology.protein
Electrophoresis, Polyacrylamide Gel
Biotechnology
Plasmids
Subjects
Details
- ISSN :
- 01681656
- Volume :
- 30
- Issue :
- 2
- Database :
- OpenAIRE
- Journal :
- Journal of biotechnology
- Accession number :
- edsair.doi.dedup.....cf056e4f01ff9dea40093369a241c918