Abstract
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.
Highlights
Signal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria
We demonstrated the first successful example of applying random and combinatorial mutagenesis on a secretory carrier to enhance total secretory target protein production in E. coli
The levels of total secreted osmotically-inducible protein Y (OsmY)-Tfu0937 produced by E. coli C41(DE3) and BL21(DE3) were first measured using the p-nitrophenyl-β-d-glucopyranoside assay to identify the more efficient strain
Summary
Signal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. Gram-positive bacteria can secrete native proteins or proteins that originate from close relatives at high y ield[2]. Their compatibility with other proteins is low and the presence of extracellular proteolytic degradation is a challenge for product quality assurance. Protein secretion in Gram-negative bacteria is less common compared to other organisms like Gram-positive bacteria, yeast and fungi. Gram-negative bacteria, like E. coli, secretes few native proteins to provide a relatively pure background for heterologous protein production. Toxic effect of some target proteins on the production host can be alleviated when the protein is secreted out of the cell
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