CAREER: Optimization of Biopharmaceutical Production by Combinatorial Cellular Engineering

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

The use of protein therapeutics for the treatment of a variety of diseases including cancer and AIDS is a rapidly expanding industry. One of the major hurdles in the development of a protein therapeutic is production of the recombinant protein at a level that can meet demand in an economically feasible manner. Present methods for the optimization of protein secretion strains involve either random mutagenesis of a host strain followed by laborious screening or ad hoc cellular manipulation. In this proposal, a novel technological platform for protein production optimization will be developed. Yeast genetics will be combined with yeast surface display technology to engineer cells for increased production of a model therapeutic protein. Yeast surface display will provide a quantitative assessment of protein expression level on a single cell basis and make the rapid screening of combinatorial libraries of yeast variants possible. A yeast gene overexpression library and gene disruption library will be screened for yeast cells having increased protein expression using flow cytometry. The functional roles of yeast cellular proteins extracted in these screens will be investigated by subcellular localization studies and by yeast two-hybrid analyses. In coordination with the research objectives, a composite classroom and laboratory course will be developed and implemented to introduce molecular and cellular level design strategies. This course sequence will train undergraduate and graduate students in modern combinatorial methods for the engineering design of RNA, DNA, proteins, and cells.