Engineering studies on the secretion of recombinant proteins from Saccharomyces cerevisiae
Abstract
The behavior of a yeast secretory model-system was experimentally characterized to determine the effect of several environmental and genetic parameters on secretion kinetics and protein localization. The secretion of cloned proteins from recombinant yeast has emerged in recent years as an important aspect of biotechnology. Secretion offers the potential to reduce separation costs in large scale fermentations, and can eliminate many of the cellular-level problems associated with the over-production of foreign proteins. Four plasmids used as a model system were transformed into the same Saccharomyces cerevisiae host, strain SEY2102. One plasmid contained the SUC2 structural gene fused to the MF$\alpha$1 promoter and signal sequence, the second contained the entire SUC2 gene, and the third and fourth contained the HLZ gene fused to the ADH1 promoter and either the MF$\alpha$1 or the rat $\alpha$-amylase signal sequence. An enzymatic fractionation method was developed and used to determine secreted product profiles in the cytoplasm, periplasm, and extracellular space of recombinant yeast cells. The response of secretion kinetics and protein localization to temperature, pH, dissolved oxygen, and substrate concentration was studied using the two invertase producing strains. The drug cycloheximide was used to determine secretion rate and the quantity of secreted invertase contained in the secretion pathway. The human lysozyme producing strains were used to study the effect of the product protein's physiochemical properties on the efficiency of secretion. Using this system a comparison of secretion efficiency was carried out between non-native, and native yeast signal sequences.
Degree
Ph.D.
Advisors
Seo, Purdue University.
Subject Area
Chemical engineering
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