Cell death caused by hyper-expression of a secretory exoglucanase in Escherichia coli

Fu, Zhibiao; Ng, Ka Lun; Lam, TL; Wong, Wan Keung

doi:10.1016/j.pep.2005.03.029

Cited by 30 publications

(21 citation statements)

References 35 publications

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“…5). This observation supports the possibility of development of a viable protocol for high-level production of precisely tailored, e.g., peptidase processed or mature, recombinant proteins, which have been shown to be detrimental or even lethal for cell growth, for example, when cells are subjected to secretory production of the proteins (Fu et al 2005). It is believed that our findings and illustrations elaborated previously and in this work may shed light on the development of versatile intein systems for protein expression.…”

Section: Discussionsupporting

confidence: 64%

Engineering versatile protein expression systems mediated by inteins in Escherichia coli

Kwong

Wong

2015

Appl Microbiol Biotechnol

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We have recently employed an intein, Saccharomyces cerevisiae vascular membrane ATPase (VMA), in conjunction with efficient expression and secretory functions formed between the ompA leader sequence and the human epidermal growth factor (EGF) gene (fused at the 5' end of VMA), and the human basic fibroblast growth factor (bFGF) gene (fused at the 3' end of VMA), to engineer an efficient intein-based Escherichia coli system for high-level co-expression of EGF and bFGF as authentic mature products. Both products were found not only excreted to the culture medium but also located, surprisingly, in the cytoplasm (Kwong and Wong 2013). In this study, we employed two structurally varied inteins, VMA and Mycobacterium xenopi GyraseA (GyrA), and further demonstrated that despite acting alone, both VMA and GyrA were able to mediate successful co-expression of two widely different proteins, EGF and an endoglucanase (Eng) in E. coli. Although EGF and Eng were initially expressed as large precursors/intermediates, they were soluble and auto-cleavable to finally yield the desired products in both the cytoplasm and culture media. The results further substantiate our postulation that the aforementioned intein/E. coli approach might lead to the development of cost-effective and versatile host systems, wherein all culture fractions are involved in producing the target proteins.

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Section: Discussionsupporting

confidence: 64%

Engineering versatile protein expression systems mediated by inteins in Escherichia coli

Kwong

Wong

2015

Appl Microbiol Biotechnol

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“…Anti-SarA and anti-SigB monoclonal antibodies were obtained as described previously (4,5). Western blot analysis was carried out on cellular lysates as described previously (11,12).…”

Section: Methodsmentioning

confidence: 99%

Overexpression of MazF _Sa in Staphylococcus aureus Induces Bacteriostasis by Selectively Targeting mRNAs for Cleavage

Tamber

Memmi

et al. 2009

J Bacteriol

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The role of chromosomally encoded toxin-antitoxin (TA) loci in bacterial physiology has been under debate, with the toxin proposed as either an inducer of bacteriostasis or a mediator of programmed cell death (PCD). We report here that ectopic expression of MazF Sa , a toxin of the TA module from Staphylococcus aureus, led to a rapid decrease in CFU counts but most cells remained viable as determined by differential Syto 9 and propidium iodide staining after MazF Sa induction. This finding suggested that the toxin MazF Sa induced cell stasis rather than cell death. We also showed that MazF Sa selectively cleaves cellular mRNAs in vivo, avoiding "important" transcripts such as recA, gyrB, and sarA mRNAs in MazF Sa -induced cells, while these three mRNAs can be cleaved in vitro. The results of Northwestern blotting showed that both sarA and recA mRNAs bind strongly to a putative RNA-binding protein. These data suggest that S. aureus likely undergoes stasis by protecting selective mRNA with RNA-binding proteins upon the expression of MazF Sa in vivo.Many bacteria have chromosomally encoded toxin-antitoxin (TA) loci in which the toxin and antitoxin genes exist in an operon and are coexpressed to form a TA complex. The toxin is stable, while the antitoxin is labile and can be degraded in vivo by host proteases (e.g., ClpP or Lon in Escherichia coli). Under conditions of stress whereby transcription of the TA operon is repressed and which hence preclude the continuous synthesis of the labile antitoxin, the more-stable toxin can unleash its toxic effect to inhibit cell growth. However, metabolic stresses, such as amino acid and carbon source starvation, have been shown to induce transcription of E. coli mazEF and other TA loci in E. coli (7,9,14). Studies with several toxin systems indicate that many toxins are probably sequence-specific endoribonucleases. For instance, MazF of E. coli cleaves mRNA at ACA sites both in vitro and in vivo (30), while the RelE toxin, also from E. coli, cleaves mRNA positioned at the ribosomal A site both in vitro and in vivo, with cleavage occurring between the second and third bases of the A site codon

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“…Thus, extracellular secretion in E. coli always correlates with non-specific leakage and cell lysis [Ni and Chen, 2009;Shokri et al, 2002], which could be due to pressure build-up in the periplasmic space. While a higher protein translocation rate is preferable, it can have lethal effects on the host cell [Fu et al, 2005].…”

Section: Introductionmentioning

confidence: 99%

Optimization of a Heterologous Signal Peptide by Site-Directed Mutagenesis for Improved Secretion of Recombinant Proteins in Escherichia coli

et al. 2012

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A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinant cyclodextrin glucanotransferase (CGTase) to the periplasmic and, eventually, extracellular space of Escherichia coli. Eight mutant SPs were constructed using site-directed mutagenesis to improve the secretion of recombinant CGTase. M5 is a mutated SP in which replacement of an isoleucine residue in the h-region to glycine created a helix-breaking or G-turn motif with decreased hydrophobicity. The mutant SP resulted in 110 and 94% increases in periplasmic and extracellular recombinant CGTase, respectively, compared to the wild-type SP at a similar level of cell lysis. The formation of intracellular inclusion bodies was also reduced, as determined by sodium dodecyl sulfate-polyacrylamyde gel electrophoresis, when this mutated SP was used. The addition of as low as 0.08% glycine at the beginning of cell growth improved cell viability of the E. coli host. Secretory production of other proteins, such as mannosidase, also showed similar improvement, as demonstrated by CGTase production, suggesting that the combination of an optimized SP and a suitable chemical additive leads to significant improvements of extracellular recombinant protein production and cell viability. These findings will be valuable for the extracellular production of recombinant proteins in E. coli.

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Cell death caused by hyper-expression of a secretory exoglucanase in Escherichia coli

Cited by 30 publications

References 35 publications

Engineering versatile protein expression systems mediated by inteins in Escherichia coli

Engineering versatile protein expression systems mediated by inteins in Escherichia coli

Overexpression of MazF _Sa in Staphylococcus aureus Induces Bacteriostasis by Selectively Targeting mRNAs for Cleavage

Optimization of a Heterologous Signal Peptide by Site-Directed Mutagenesis for Improved Secretion of Recombinant Proteins in Escherichia coli

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Cell death caused by hyper-expression of a secretory exoglucanase in Escherichia coli

Cited by 30 publications

References 35 publications

Engineering versatile protein expression systems mediated by inteins in Escherichia coli

Engineering versatile protein expression systems mediated by inteins in Escherichia coli

Overexpression of MazF Sa in Staphylococcus aureus Induces Bacteriostasis by Selectively Targeting mRNAs for Cleavage

Optimization of a Heterologous Signal Peptide by Site-Directed Mutagenesis for Improved Secretion of Recombinant Proteins in Escherichia coli

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Overexpression of MazF _Sa in Staphylococcus aureus Induces Bacteriostasis by Selectively Targeting mRNAs for Cleavage