THESIS
2011
xiv, 92 p. : ill. ; 30 cm
Abstract
Production of recombinant proteins by secretory hyper-expression has many advantages over
intracellular 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 cou...[
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Production of recombinant proteins by secretory hyper-expression has many advantages over
intracellular 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 PspAΔ
25 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.
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