Flavin-Based Fluorescent Protein EcFbFP Auto-Guided Surface Display of Methyl Parathion Hydrolase in Escherichia coli
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Methyl parathion hydrolase (MPH) plays an important role in degrading a range of organophosphorus compounds. In order to display MPH on the cell surface of Escherichia coli strain RosettaBlue™, the Flavin-based fluorescent protein EcFbFP was severed as an auto-anchoring matrix. With net negative charges of EcFbFP supplying the driving forces, fusion protein MPH-EcFbFP through a two-step auto-surface display process was finally verified by (a) inner membrane translocation and (b) anchoring at outer membrane. Cells with surface-displayed MPH obtained activity of 0.12 U/OD600 against substrate methyl parathion. MPH when fused with engineered EcFbFP containing 20 net negative charges exhibited fivefold higher anchoring efficiency and tenfold higher enzymatic catalytic activity of 1.10 U/OD600. The above result showed that MPH was successfully displayed on cell surface and can be used for biodegradation of methyl parathion.
KeywordsFlavin-based fluorescent protein EcFbFP Cell-surface display Net negative charge Methyl parathion hydrolase
Methyl parathion hydrolase
Escherichia coli codon-optimized FMN-based fluorescent protein
EcFbFP mutant with 0 net charge
EcFbFP mutant with 2 net positive charges
EcFbFP mutant with 4 net positive charges
EcFbFP mutant with 20 net negative charges
MPH fused with EcFbFP
MPH fused with EcFbFP(-20)
Super folder green fluorescent protein
MCherry fluorescent protein
MPH fused with sfGFP
MPH fused with mCherry
The authors would like to thank professor Li Yi in Hubei University for the critical revision of the manuscript, thank Dr. Mohanty P.B. in University of Oklahoma (U.S.A.) for the analysis of protein structure.
L.B., Z.Z., and L.M. conceived, designed, and coordinated the study, and wrote the paper. R.T. performed the experiments shown in Figs. 1, 2, 3, 4, and 5. L.B. performed tables, and contributed to paper writing. W.S. performed the experiments shown in Fig. 5.
This work was supported by Foundation for National Key Basic Research Program of China (No. 2013CB910801 to L.M.), National Science Foundation of Hubei Province (No. 2013CFA133 to L.M.), High and New Technology Industrial Innovative Research Groups of the Wuhan Science and technology Bureau’s department (No. 2014CFA126, to L.M.).
Compliance with Ethical Standards
Conflict of interest
The authors declared that they have no conflicts of interest with the contents of this article.
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