An integrating vector for tunable, high copy, stable integration into the dispersed Ty delta sites of Saccharomyces cerevisiae.
We have constructed a yeast integration vector targeted to chromosomal Ty delta sequences and used it to create Saccharomyces cerevisiae strains with stable tandem integrations ranging from 1 to 30 vector copies. The vector carries the bacterial NEO gene, allowing copy number to be tuned by varying G418 resistance, which generally increases with copy number as determined by quantitative Southern blot. Tandem integration into a single site is most commonly observed, but single-copy and two-site integration is also observed. Bovine pancreatic trypsin inhibitor was constitutively expressed and secreted using the NEO-based delta vector, and secretion levels were 2-10-fold improved relative to commonly used 2 mu multicopy yeast plasmids. The NEO-based Ty delta vector is a powerful tool for stable heterologous protein expression and secretion in yeast.
Parekh
R N
RN
Department of Chemical Engineering, University of Illinois, Urbana 61801, USA.
Shaw
M R
MR
Wittrup
K D
KD
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002417:Cattle; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics*; D003470:Culture Media; D005784:Gene Amplification; D015870:Gene Expression; D005822:Genetic Vectors; D007613:Kanamycin Resistance / Q000235:genetics; D018598:Minisatellite Repeats; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000096:biosynthesis / Q000235:genetics
D003470:Culture Media; D014359:Trypsin Inhibitor, Kazal Pancreatic