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Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli.
Gonzalez-Perez, David; Ratcliffe, James; Tan, Shu Khan; Wong, Mary Chen May; Yee, Yi Pei; Nyabadza, Natsai; Xu, Jian-He; Wong, Tuck Seng; Tee, Kang Lan.
Afiliación
  • Gonzalez-Perez D; Department of Chemical and Biological Engineering, The University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD, UK.
  • Ratcliffe J; Department of Drug Discovery, Moffitt Cancer Center & Research Institute, Stabile Research Building, 12902 Magnolia Dr, Tampa, FL, 33612, USA.
  • Tan SK; Department of Chemical and Biological Engineering, The University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD, UK.
  • Wong MCM; Department of Chemical and Biological Engineering, The University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD, UK.
  • Yee YP; Department of Chemical and Biological Engineering, The University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD, UK.
  • Nyabadza N; Department of Chemical and Biological Engineering, The University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD, UK.
  • Xu JH; Department of Chemical and Biological Engineering, The University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD, UK.
  • Wong TS; Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
  • Tee KL; Department of Chemical and Biological Engineering, The University of Sheffield, Sir Robert Hadfield Building, Mappin Street, Sheffield, S1 3JD, UK. t.wong@sheffield.ac.uk.
Sci Rep ; 11(1): 5290, 2021 03 05.
Article en En | MEDLINE | ID: mdl-33674702
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 ß-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.
Asunto(s)

Texto completo: 1 Base de datos: MEDLINE Asunto principal: Mutagénesis / Proteínas de Escherichia coli / Proteínas de Unión Periplasmáticas / Escherichia coli / Vías Secretoras / Sistemas de Secreción Bacterianos Tipo de estudio: Clinical_trials Idioma: En Revista: Sci Rep Año: 2021 Tipo del documento: Article

Texto completo: 1 Base de datos: MEDLINE Asunto principal: Mutagénesis / Proteínas de Escherichia coli / Proteínas de Unión Periplasmáticas / Escherichia coli / Vías Secretoras / Sistemas de Secreción Bacterianos Tipo de estudio: Clinical_trials Idioma: En Revista: Sci Rep Año: 2021 Tipo del documento: Article