title: Anchorage of GFP fusion on the cell surface of Pseudomonas putida.
abstract: Here we report the cell surface display of organophosphorus hydrolase (OPH) and green fluorescent protein (GFP) fusion by employing the N- and C-terminal domains of ice nucleation protein (INPNC) as an anchoring motif. An E. coli-Pseudomonas shuttle vector, pNOG33, coding for INPNC-OPH-GFP was constructed for targeting the fusion onto the cell surface of p-nitrophenol (PNP)-degrading P. putida JS444. The surface localization of INPNC-OPH-GFP was verified by cell fractionation, Western blot, proteinase accessibility, and immunofluorescence microscopy. Furthermore, the functionality of the surface-exposed OPH-GFP was demonstrated by OPH assays and fluorescence measurements. Surface display of macromolecular OPH-GFP fusion (63 kDa) neither inhibited cell growth nor affected cell viability. These results suggest that INP is an useful tool for the presentation of heterologous proteins on cell surfaces of indigenous microbes. The engineered P. putida JS444 degraded organophosphates (OPs) as well as PNP rapidly and could be easily monitored by fluorescence. Parathion (100 mg kg⁻¹) could be degraded completely within 15 days in soil inoculated with the engineered strain. These merits make this engineered strain an ideal biocatalyst for in situ bioremediation of OP-contaminated soil.
authors.affiliation: Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
authors.forename: Yulan
authors.initials: Y
authors.lastname: Yuan
authors.forename: Chao
authors.initials: C
authors.lastname: Yang
authors.forename: Cunjiang
authors.initials: C
authors.lastname: Song
authors.forename: Hong
authors.initials: H
authors.lastname: Jiang
authors.forename: Ashok
authors.initials: A
authors.lastname: Mulchandani
authors.forename: Chuanling
authors.initials: C
authors.lastname: Qiao
mesh_terms: D043303:Aryldialkylphosphatase / Q000235:genetics / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001673:Biodegradation, Environmental; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D009596:Nitrophenols / Q000378:metabolism; D015202:Protein Engineering; D021381:Protein Transport; D011549:Pseudomonas / Q000201:enzymology; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D009596:Nitrophenols; D011993:Recombinant Fusion Proteins; C063113:ice nucleation protein; D049452:Green Fluorescent Proteins; D043303:Aryldialkylphosphatase; C024836:4-nitrophenol