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Wittrup, K. Dane Shaw, Mark Robert 2014-12-15T19:54:47Z 2014-12-15T19:54:47Z 10000-01-01 1995 1995 Gene amplification plays a central role in maximizing yield for protein production. Generally, more copies of a gene generally lead to an increase in protein production. The 2$\mu$ plasmid of Saccharomyces cerevisiae has been used extensively as a vector for foreign protein expression because of its natural amplification to 60-100 copies per cell. Integration of genes into the chromosomes provides the advantage of increasing stability over 2$\mu$ based vectors. Integration vectors allow integration of multiple copies of a gene leading to improved protein production. The amplification of the 2$\mu$ plasmid occurs through a recombination mechanism which I have simulated using two free parameters: the FLP recombinase DNA-binding probability and enzymatic activity. Model results indicate that a single plasmid amplifies to 5.5-7 copies on average at maximal FLP concentrations. The model predicts the existence of topologically novel replication intermediates. I have constructed a yeast integration vector targeted to chromosomal Ty $\delta$ sequences and used it to create strains with stable tandem integrations ranging from 1 to 30 vector copies. The vector carries the bacterial NEO gene, conferring G418 resistance, which generally increases with copy number as determined by Southern blot. Bovine pancreatic trypsin inhibitor was expressed and secreted using the NEO-based $\delta$ vector. Expression levels of BPTI mRNA were measured and correlated with the amount of secreted protein. Quantitative Western blots were performed to measure the amount of unfolded protein accumulating inside the cell. The relationship between the amount of secreted BPTI, the expression level of mRNA and the accumulation of unfolded protein support the hypothesis that the secretory pathway of yeast can be saturated and that protein folding is the rate-limiting step in secretion. This implies that an optimum copy number exists for maximum protein production at a point where the rate of protein synthesis is balanced by the rate of protein folding and secretion. The NEO-based Ty $\delta$ vector is a powerful tool for identifying this optimum for foreign proteins as well as for stable expression and secretion of heterologous proteins in yeast. Made available in DSpace on 2014-12-15T19:54:47Z (GMT). No. of bitstreams: 1 9624492.pdf: 4768597 bytes, checksum: d89ad3eb7242a13df8c534e3ac2e0d1d (MD5) Previous issue date: 1995 Embargo set by: Seth Robbins for item 69966 Lift date: Forever Reason: Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs Restricted to the U of I community idenfinitely during batch ingest of legacy ETDs U of I Only 115 p. Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1995. http://hdl.handle.net/2142/69800 (UMI)AAI9624492 Biology, Molecular Biology, Genetics Engineering, Chemical Gene Amplification for the Improved Secretion of Foreign Proteins in the Yeast Saccharomyces Cerevisiae text Chemical Engineering Chemical Engineering University of Illinois at Urbana-Champaign Dissertation Ph.D.