Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli
| Autor: | Shu Khan Tan, Tuck Seng Wong, Jian-He Xu, Mary Chen May Wong, Yi Pei Yee, James Ratcliffe, David Gonzalez-Perez, Kang Lan Tee, Natsai Nyabadza |
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| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Signal peptide Expression systems Science Recombinant Fusion Proteins 030106 microbiology Mutagenesis (molecular biology technique) Protein Sorting Signals medicine.disease_cause Article Green fluorescent protein 03 medical and health sciences Mutation Rate Escherichia coli medicine Secretion Bacterial Secretion Systems Synthetic biology Secretory Pathway Multidisciplinary Chemistry Escherichia coli Proteins beta-Glucosidase Temperature Lipase Directed evolution Thermobifida Luminescent Proteins Protein Transport 030104 developmental biology Secretory protein Biochemistry Mutagenesis Periplasmic Binding Proteins Medicine Target protein Microorganisms Genetically-Modified Bacillus subtilis |
| Zdroj: | Scientific Reports Scientific Reports, Vol 11, Iss 1, Pp 1-13 (2021) |
| ISSN: | 2045-2322 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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