OneClick: A Program for Designing Focused Mutagenesis Experiments

Warburton, Mark; Ali, Hossam Omar; Liong, Wai Choon; Othusitse, Arona Martin; Zubir, Amir Zaki Abdullah; Maddock, Steve; Wong, Tuck Seng

doi:10.3934/bioeng.2015.3.126

Cited by 6 publications

(7 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Mutagenic primers (Table 1), PCR mixtures and PCR conditions for all site-directed mutagenesis and saturation mutagenesis studies were designed using OneClick programme, which is publicly accessible via the web-link: http://tucksengwong.staff.shef.ac.uk/OneClick/ (Warburton et al 2015).…”

Section: Methodsmentioning

confidence: 99%

Accelerated directed evolution of dye-decolorizing peroxidase using a bacterial extracellular protein secretion system (BENNY)

Alessa

Tee

González-Pérez

et al. 2019

Bioresour. Bioprocess.

View full text Add to dashboard Cite

Background Dye-decolorizing peroxidases (DyPs) are haem-containing peroxidases that show great promises in industrial biocatalysis and lignocellulosic degradation. Through the use of Escherichia coli osmotically-inducible protein Y (OsmY) as a bacterial extracellular protein secretion system (BENNY), we successfully developed a streamlined directed evolution workflow to accelerate the protein engineering of DyP4 from Pleurotus ostreatus strain PC15. Result After 3 rounds of random mutagenesis with error-prone polymerase chain reaction (epPCR) and 1 round of saturation mutagenesis, we obtained 4D4 variant (I56V, K109R, N227S and N312S) that displays multiple desirable phenotypes, including higher protein yield and secretion, higher specific activity (2.7-fold improvement in k cat / K m ) and higher H 2 O 2 tolerance (sevenfold improvement based on IC 50 ). Conclusion To our best knowledge, this is the first report of applying OsmY to simplify the directed evolution workflow and to direct the extracellular secretion of a haem protein such as DyP4. Electronic supplementary material The online version of this article (10.1186/s40643-019-0255-7) contains supplementary material, which is available to authorized users.

show abstract

Section: Methodsmentioning

confidence: 99%

Accelerated directed evolution of dye-decolorizing peroxidase using a bacterial extracellular protein secretion system (BENNY)

Alessa

Tee

González-Pérez

et al. 2019

Bioresour. Bioprocess.

View full text Add to dashboard Cite

show abstract

“…Site-directed mutagenesis experiments were performed using partially overlapping primers designed by the OneClick program 33 . Primers OsmY(L6P)-FP and OsmY(L6P)-RP were used for OsmY(L6P), primers OsmY(V154R)-FP and OsmY(S154R)-RP were used for OsmY(S154R), and primers OsmY(V191E)-FP and OsmY(V191E)-RP were used for OsmY(V191E) (Supplementary Table S1 ).…”

Section: Methodsmentioning

confidence: 99%

Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

González-Pérez

Tan

et al. 2021

Sci Rep

View full text Add to dashboard Cite

Signal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli.

show abstract

“…Mutagenesis was performed on the pET24a vector containing the HLTEV WT‐XCL1 fusion protein using pairs of complementary primers (Supplementary S1) and the QuikChange II site‐directed mutagenesis kit (Agilent) to create W55D and CC3 (V21C, V29C) mutants (Warburton, Omar Ali, & Choon Liong, 2015). The resulting plasmid was transformed into Escherichia coli strain BL21(DE3) (New England Biolab) and overexpression induced using auto‐induction media (Studier, 2014) at 30°C for 24‐hr.…”

Section: Methodsmentioning

confidence: 99%

The chemokine lymphotactin and its recombinant variants in oral cancer cell regulation

et al. 2020

View full text Add to dashboard Cite

show abstract

OneClick: A Program for Designing Focused Mutagenesis Experiments

Cited by 6 publications

References 25 publications

Accelerated directed evolution of dye-decolorizing peroxidase using a bacterial extracellular protein secretion system (BENNY)

Accelerated directed evolution of dye-decolorizing peroxidase using a bacterial extracellular protein secretion system (BENNY)

Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

The chemokine lymphotactin and its recombinant variants in oral cancer cell regulation

Contact Info

Product

Resources

About