Surface Display of Organophosphorus Hydrolase on E. coli Using N-Terminal Domain of Ice Nucleation Protein InaV

2012 ◽  
Vol 22 (2) ◽  
pp. 234-238 ◽  
Author(s):  
Samaneh Khodi
Keyword(s):  
Surface Display ◽  
Ice Nucleation ◽  
Organophosphorus Hydrolase ◽  
Ice Nucleation Protein ◽  
E Coli ◽  
Terminal Domain

scholarly journals Cotranslocation of Methyl Parathion Hydrolase to the Periplasm and of Organophosphorus Hydrolase to the Cell Surface of Escherichia coli by the Tat Pathway and Ice Nucleation Protein Display System

2009 ◽  
Vol 76 (2) ◽  
pp. 434-440 ◽  
Author(s):  
Chao Yang ◽  
Roland Freudl ◽  
Chuanling Qiao ◽  
Ashok Mulchandani
Keyword(s):  
Escherichia Coli ◽  
Methyl Parathion ◽  
Ice Nucleation ◽  
Organophosphorus Hydrolase ◽  
Display System ◽  
Ice Nucleation Protein ◽  
Methyl Parathion Hydrolase ◽  
E Coli ◽  
Tat Pathway ◽  
Parathion Hydrolase

ABSTRACT A genetically engineered Escherichia coli strain coexpressing organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH) was constructed for the first time by cotransforming two compatible plasmids. Since these two enzymes have different substrate specificities, the coexpression strain showed a broader substrate range than strains expressing either one of the hydrolases. To reduce the mass transport limitation of organophosphates (OPs) across the cell membrane, MPH and OPH were simultaneously translocated to the periplasm and cell surface of E. coli, respectively, by employing the twin-arginine translocation (Tat) pathway and ice nucleation protein (INP) display system. The resulting recombinant strain showed sixfold-higher whole-cell activity than the control strain expressing cytosolic OP hydrolases. The correct localization of MPH and OPH was demonstrated by cell fractionation, immunoblotting, and enzyme activity assays. No growth inhibition was observed for the recombinant E. coli strain, and suspended cultures retained almost 100% of the activity over a period of 2 weeks. Owing to its high level of activity and superior stability, the recombinant E. coli strain could be employed as a whole-cell biocatalyst for detoxification of OPs. This strategy of utilizing dual translocation pathways should open up new avenues for cotranslocating multiple functional moieties to different extracytosolic compartments of a bacterial cell.

scholarly journals Cell Surface Display of Human Immunodeficiency Virus Type 1 gp120 on Escherichia coli by Using Ice Nucleation Protein

1999 ◽  
Vol 6 (4) ◽  
pp. 499-503 ◽  
Author(s):  
Young-Don Kwak ◽  
Seung-Ku Yoo ◽  
Eui-Joong Kim
Keyword(s):  
Escherichia Coli ◽  
Human Immunodeficiency Virus ◽  
Surface Display ◽  
Cell Surface Display ◽  
Viral Proteins ◽  
Ice Nucleation ◽  
Ice Nucleation Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT A new system designed for cell surface display of recombinant proteins on Escherichia coli has been evaluated for expression of eukaryotic viral proteins. Human immunodeficiency virus type 1 (HIV-1) gp120 was fused to the C terminus of ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. Western blotting, immunofluorescence microscopy, fluorescence-activated cell-sorting analysis, whole-cell enzyme-linked immunosorbent assay, and ice nucleation activity assay confirmed the successful expression of HIV-1 gp120 on the surface ofEscherichia coli. This study shows that the INP system can be used for the expression of eukaryotic viral proteins. There is also a possibility that the INP system can be used as an AIDS diagnostic system, an oral vaccine delivery system, and an expression system for various heterologous higher-molecular-weight proteins.

Sign in / Sign up

Export Citation Format

Share Document