Enhancement of secretory hyper-expression of heterologous proteins in escherichia coli

Abstract

Production of recombinant proteins by secretory hyper-expression has many advantages overintracellular expression in Escherichia coli. However, saturated translocation of heterologous proteins through the SecYEG pathway might cause severe cell death. A threshold ratio (Critical Value, CV) of pre-mature protein to mature protein in the lysate was previously formulated to correlate with host cell viability to enable maximum secretory production of heterologous proteins. In this study, based on the guidance of CV, we demonstrated the use of four efficient B. subtilis promoters, vegG P and its three variants, which exhibited different transcriptional strengths to furnish graded expression of a Cellulomonas fimi endoglucanase (Eng) in E. coli. A practical approach of viable cell counts was established to predict the maximum tolerable level of secretory Eng, thereby enabling a fast access to a secretory level of Eng that was close to its CV. Furthermore, employing a C. fimi exoglucanase (Exg) as a model, it was shown that its CV, which identified the tolerable limit of secretory production of Exg, might be largely increased from a ratio of 20/80 to 45/55 upon co-expression of the phage shock protein A (PspA). Using the E. coli (pM1VegGCexLpspA) co-expression system, after induced expression of Exg and PspA with IPTG and L-arabinose, respectively, the Exg activity detected in the culture medium was 1.65 U/ml, which was 80 % higher than the best yield ever reported for this protein. However, co-expression with a truncated mutant PspAΔ25 failed to give any detectable improvement. Results from flow-cytometric analysis supported the conclusion that the co-expressed PspA helped defer membrane depolarization and damage due to hyper-secretion of heterologous proteins in E. coli. In addition to the illustration of co-expressing PspA to enhance secretory production of Exg in E. coli, our findings enhance our understanding of stress response triggered by hyper-expression of secretory heterologous proteins in E. coli. The findings may also offer a general approach for the development of potent E. coli strains for efficient secretory/excretory production of heterologous proteins.

Date of Award	2011
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology