An Integrating Vector for Tunable, High Copy, Stable Integration into the Dispersed Ty δ Sites of Saccharomyces cerevisiae

Abstract

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.

High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor

“…The multi-integrating pITy4-wt plasmid (Parekh et al, 1996), previously used for overexpression of A 2 aR in S. cerevisiae, was also employed in these studies. pITy contains a Gal1-10 promotor for inducible expression upon addition of galactose in the growth medium, a synthetic pre-pro leader sequence which directs the protein for secretion (Clements et al, 1991;Parekh et al, 1995), and the yeast alpha terminator.…”

“…After transformation, cells were screened for their ability to express either A 2 aR-His 10 or A 2 aR-GFP-His 10 , since the pITy vector is capable of integrating 1-30 times within different locations of the yeast chromosome (Parekh et al, 1996). Transformed BJ5464 containing integrated A 2 aR-His 10 DNA were selected from individual colonies, grown to saturation, and expression was induced.…”

“…Since the pITy integrating vector is capable of integrating between 1-50 copies within the yeast chromosome, and at varying location (Parekh et al, 1996), screening of transformed cells was necessary to achieve adequate GPCR expression levels. Previously, flow cytometry was successfully implemented to isolate a high-expressing A 2 aR-GFP yeast cell (~4 mg/L of culture active protein) from a population of transformed cells (Niebauer et al, 2004).…”

“…The ability to isolate a large amount of high-expressing clones is attributed to selection on 2.0 mg/mL G418 plates, a condition which is stringent enough to select primarily for cells which have integrated multiple copies of the DNA in suitable locations within the yeast genome. High concentration G418 selection has been previously correlated to increased pITy integration and expression of bovine pancreatic trypsin inhibitor in S. cerevisiae (Parekh et al, 1996).…”

Pressure response of protein backbone structure. Pressure‐induced amide 15N chemical shifts in BPTI

“…Yeast strain RDG194 is a gift from Dr. K. Wittrup~Parekh et al, 1996;Kowalski et al, 1998!. To express uniformly 15 N-labeled EA-BPTI, 10 mL YPD~2% bacto-yeast extract, 1% bacto-peptone, 2% dextrose!…”