To summarize, a variety of stable vectors and efficient promoters and secretion signals are available in yeast for engineering the secretion of any protein of interest. Since secretion is growth-associated, we have favored the use of constitutive promoters and moderate copy number integrated vectors. This is because (1) heterologous gene expression from very high copy number vectors is frequently deleterious to growth and (2) delaying gene expression until after the most rapid cell growth phase is cumbersome on a large scale. Methods are available for dividing the total process into growth and production/secretion phases, but they appear worthwhile only when expression of the engineered protein compromises growth significantly. Even with these useful tools, it is frequently helpful to enlist the aid of mutant host strains in order to maximize secretion of a desired protein. Mutations in the PMR1 gene have proved effective in a number of different cases. Moreover, it is possible to identify new host strains tailored to specific needs by applying activity screens to mutagenized colonies growing on petri plates. Finally, colony screens such as the ones described here for active secreted enzymes are useful for routine strain construction. For example, they may be applied to identify the most productive strain from a large number of clones following a transformation or genetic cross. In addition, these screens may be used for characterizing the products of random mutagenesis of the gene encoding the secreted enzyme. The resulting structure-function information can be used to identify regions of the enzyme involved in different activities and to build new enzymes with different characteristics.

• PMID: 2005805
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Reference Type

Journal Article | Research Support, Non-U.S. Gov't

Authors

Moir DT, Davidow LS

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