Use of Amino Acids as Inducers for High-Level Protein Expression in the Single-Protein Production System

Vaiphei, S. Thangminlal; Mao, Lili; Shimazu, Tsutomu; Park, Jung‐Ho; Inouye, Masayori

doi:10.1128/aem.02138-10

Cited by 3 publications

(4 citation statements)

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“…The influence of IPTG on protein induction found to be confirming the reports that low concentration of IPTG supports heterologous IMP expression [35], [37]. Negative effects of IPTG when interacting with certain gene products was reported as the reason for low expression of cloned genes [16].…”

Section: Discussionsupporting

confidence: 84%

Expression of Leptospira membrane proteins Signal Peptidase (SP) and Leptospira Endostatin like A (Len A) in BL-21(DE3) is toxic to the host cells

Satheeshkumar

Anu

et al. 2018

Journal of Genetic Engineering and Biotechnology

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Heterologous expression of Integral Membrane Proteins (IMPs) is reported to be toxic to the host system in many studies. Even though there are reports on various concerns like transformation efficiency, growth properties, protein toxicity, inefficient expression and protein degradation in IMP overexpression, no studies so far addressed these issues in a comprehensive way. In the present study, two transmembrane proteins of the pathogen Leptospira interrogans , namely Signal peptidase (SP), and Leptospira Endostatin like A (Len-A) were taken along with a cytosolic protein Hydrolase (HYD) to assess the differences in transformation efficiency, protein toxicity, and protein stability when over expressed in Escherichia coli ( E. coli ). Bioinformatics analysis to predict the transmembrane localization indicated that both SP and Len are targeted to the membrane. The three proteins were expressed in full length in the E. coli expression strain, BL 21 (DE3). Significant changes were observed for the strains transformed with IMP genes under the parameters analysed such as, the transformation efficiency, survival of colonies on IPTG-plate, culture growth kinetics and protein expression compared to the strain harbouring the cytosolic protein gene.

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Section: Discussionsupporting

confidence: 84%

Expression of Leptospira membrane proteins Signal Peptidase (SP) and Leptospira Endostatin like A (Len A) in BL-21(DE3) is toxic to the host cells

Satheeshkumar

Anu

et al. 2018

Journal of Genetic Engineering and Biotechnology

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“…Subsequent addition of the amino acid induces exclusive production of the recombinant protein, since production of host proteins is blocked by the toxic action of MazF. Therefore, this approach allows not only single protein production but also high-efficiency isotope-labelling of the target protein (Vaiphei et al, 2010).…”

Section: Class II Ta Systems As Biotechnological Toolsmentioning

confidence: 99%

Prokaryotic toxin-antitoxin systems--the role in bacterial physiology and application in molecular biology.

Bukowski

Rojowska²,

Władyka³

2011

Acta Biochim Pol

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Bacteria have developed multiple complex mechanisms ensuring an adequate response to environmental changes. In this context, bacterial cell division and growth are subject to strict control to ensure metabolic balance and cell survival. A plethora of studies cast light on toxin-antitoxin (TA) systems as metabolism regulators acting in response to environmental stress conditions. Many of those studies suggest direct relations between the TA systems and the pathogenic potential or antibiotic resistance of relevant bacteria. Other studies point out that TA systems play a significant role in ensuring stability of mobile genetic material. The evolutionary origin and relations between various TA systems are still a subject of a debate. The impact of toxin-antitoxin systems on bacteria physiology prompted their application in molecular biology as tools allowing cloning of some hard-to-maintain genes, plasmid maintenance and production of recombinant proteins.

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“…The toxin MazF as an endoribonuclease cleaves single‐stranded RNA at the “ACA” recognition sequence whereas MazE is the cognate antitoxin for neutralization of MazF toxicity (Y. Zhang et al, ). Inspired by the action mechanism, MazF has been recruited for construction of a condensed single protein production system (Suzuki, Mao, & Inouye, ; Vaiphei, Mao, Shimazu, Park, & Inouye, ). In addition, MazF has also been used as a novel counter‐selection marker for genome engineering in several organisms (Song et al, ; J. J. Yang, Jiang, & Yang, ; X.…”

Section: Introductionmentioning

confidence: 99%

“…Zhang et al, 2003). Inspired by the action mechanism, MazF has been recruited for construction of a condensed single protein production system (Suzuki, Mao, & Inouye, 2007;Vaiphei, Mao, Shimazu, Park, & Inouye, 2010). In addition, MazF has also been used as a novel counter-selection marker for genome engineering in several organisms (Song et al, 2015;J.…”

mentioning

confidence: 99%

A new sRNA‐mediated posttranscriptional regulation system for Bacillus subtilis

Yang

Wei

Liu

et al. 2018

Biotech & Bioengineering

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Many genetic tools for gene regulation have been developed during the past decades. Some of them edit genomic DNA, such as nucleotides deletions and insertions, while the others interfere with the gene transcriptions or messenger RNA translation. Here, we report a posttranscriptional regulation tool which is termed "Modulation via the small RNA (sRNA)-dependent operation system: MS-DOS" by engineering the type I toxin-antitoxin system in Bacillus subtilis. MS-DOS depends simply on insertion of an operation region (OPR; partial toxin-encoding region) downstream of a genomic open reading frame of interest and overexpression of the coupling antitoxin sRNA from a plasmid. Pairing between the OPR and the sRNA will trigger the RNAse degradation of the transcripts of selected genes. MS-DOS allows for the quantitative, specific, and reversible knockdown of single or multiple genomic genes in B. subtilis. We also showed that the truncated antitoxin SR4 with 53 nt length is sufficient to repress gene expression. Superior to other existing RNA based interfering systems, MS-DOS allows simultaneous knockdown of multiple genes with effortless expression of a single antitoxin RNA. This sRNA-guided repression system will further enrich the gene regulation tools and expand the gene regulation flexibility.

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Use of Amino Acids as Inducers for High-Level Protein Expression in the Single-Protein Production System

Cited by 3 publications

References 0 publications

Expression of Leptospira membrane proteins Signal Peptidase (SP) and Leptospira Endostatin like A (Len A) in BL-21(DE3) is toxic to the host cells

Expression of Leptospira membrane proteins Signal Peptidase (SP) and Leptospira Endostatin like A (Len A) in BL-21(DE3) is toxic to the host cells

Prokaryotic toxin-antitoxin systems--the role in bacterial physiology and application in molecular biology.

A new sRNA‐mediated posttranscriptional regulation system for Bacillus subtilis

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