9634036 Georgiou This proposal is on research to improve the production of correctly folded complex eucaryotic protein in E. coli. It is proposed to employ thrombolytic enzyme tissue plasminogen activator (tPA) as the model protein for optimizing the expression of secreted multidisulfide proteins in bacteria. It is expected that conditions which maximize the formation of the correct disulfide bonds in tPA will also be useful for the expression of simpler proteins with fewer disulfide bonds. A three part strategy is proposed. This includes: (1) Cellular Engineering: This approach utilizes a recent discovery by the investigators that the mammalian protein disulfide isomerase (PDI) can be expressed in a functional form in E. coli where it can mediate the expression of low, but significant, amounts of active tPA. Conditions that enhance the activity of PDI in the bacterial periplasmic space, and thus result in increased yields of correctly folded tPA, will be determined. (2) Genetic Screening: The use of genetic screens to isolate mutants producing high amounts of correctly folded tPA will be employed since disulfide bond formation is a complex process that may prove difficult to optimize solely by cell engineering approaches. Mutants exhibiting the highest titer of active tPA will be isolated and characterized to determine the genetic lesions that result in more efficient disulfide bond formation. (3) Fermentation Engineering: A systematic study of the effect of fermentation conditions which are known to have an influence on tPA folding will be conducted. These include parameters such as dissolved oxygen concentration, the redox potential of the growth medium, the presence of low molecular weight thiols and disulfides and culture pH. The in vivo oxygen states of the enzymes that catalyze disulfide bond formation will be determined so that the effect of growth conditions on function of the disulfide machinery can be understood. Also, fermentation c onditions that maximize cell density while maintaining the optimal oxidation state for heterologous protein folding will be investigated. This research is a cooperative activity with Genentech Inc. in South San Francisco, California. Genentech will commit significant resources including scientists' time fermentation equipment, expression vectors, antibodies, analytical support, tPA samples etc. towards the completion of this research. ***
