Enhance Heterologous Protein Production in High-Density Plant Suspension Cultures using Cell-Cycle Engineering and GFP-based On-line Optimization

University Of Hawaii, Honolulu

Investigators

Abstract

The overall goal of this project is to develop plant cell bioreactor cultures into a more effective system for producing high-value recombinant proteins. The specific objectives of the present research are as follows: 1) Test inducible cell-cycle control to maximize the useful culture life and yield of the target protein; 2) Employ tested molecular strategies that complement the cell-cycle control to further enhance the level of target protein expression; 3) Optimize protein production using on-line GFP-fusion monitoring and dynamic process optimization in high-density cultures. To accomplish these objectives, the Principal Investigators (PIs) will embark on the following research tasks: (1) examine the effect of expressing a cell-cycle inhibitor on cell proliferation in plant suspension cultures; (2) study coordinated expression of the target gene and the cell-cycle gene to enhance the target protein production; (3) exploit suitable targeting and endoplasmic reticulum (ER)-retention signals to promote secretion and to stabilize the target protein; (4) engineer GFP as a dual function fusion tag for process monitoring and protein purification; (5) investigate signal processing of on-line detected fluorescence of GFP-fusion proteins; (6) devise an effective dynamic process optimization scheme that draws on the on-line fluorescence data; and (7) develop high-density perfusion and fed-batch cultures integrated with the controlled proliferation technology and the fusion protein monitoring/optimization schemes. Tobacco will be used as the model host system. Human granulocyte macrophage colony-stimulating factor (GMCSF) with GFP fusion is chosen as a model protein in this research. The proposed research should lead to development of a more effective plant cell culture process for recombinant protein production. It should also shed light on regulation of the plant cell cycle and cell-cycle dependence of foreign gene expression in plant cells.