Expression of Single Chain Antibodies (ScFvs) for c‐myc Oncoprotein in Recombinant Escherichiacoli Membranes by Using the Ice‐Nucleation Protein of Pseudomonassyringae

Bassi, Amarjeet; Ding, Daniel N.; Gloor, Gregory B.; Margaritis, Argyrios

doi:10.1021/bp000053k

Cited by 17 publications

(11 citation statements)

References 33 publications

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“…It is membrane-anchored via the glycosylphosphatidylinositol (GPI)-anchor sequence, and this is quite unique for a prokaryote since this motif is normally found only in eukaryotic cells (23). By fusing the gene of interest to the C-terminus of INP and expressing this structure in E. coli, various active enzymes (18, 19, 24, 25) and single-chain antibodies (26) have been successfully anchored to the cell surface. Our results in this paper demonstrate that CaE B1 can be similarly expressed on the cell surface with good stability using the truncated version of the INP protein (INPNC).…”

Section: Discussionmentioning

confidence: 99%

Bioremediation of Organophosphorus Pesticides by Surface-Expressed Carboxylesterase from Mosquito on Escherichia Coli

et al. 2004

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The insecticide resistance-associated esterase, carboxylesterase B1 (CaE B1), from mosquito was used to degrade the organophosphorus compounds. To eradicate the need for enzyme purification and minimize the resistance to mass transport of the substrate and product across the cell membranes, the CaE B1 was displayed on the cell surface of Escherichia coli fused to the C-terminus of the ice nucleation protein (INP). The presence of CaE B1 on the bacterial cell surface was verified by SDS-PAGE, Western blotting analysis, and immunofluorescence microscopy. More than 50% of active CaE B1 is exported across the membrane and anchored onto the cell surface as determined by proteinase accessibility and cell fractionation experiments. In contrast, only a 6% drop in activity for proteinase K-treated cells was detected from E.coli cells containing pET-B1. From the degradation experiment, more than 80% of the malathion was degraded by whole cells containing plasmid pUC-NC-B1. Constitutive expression of CaE B1 on the surface using INPNC resulted in no cell lysis, and the suspended cultures also exhibited good stability. Because of their high biodegradation activity and superior stability, these "live biocatalysts" are promising for detoxification of organophosphorus pesticides.

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Section: Discussionmentioning

confidence: 99%

Bioremediation of Organophosphorus Pesticides by Surface-Expressed Carboxylesterase from Mosquito on Escherichia Coli

et al. 2004

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“…The artificial protein retains the specificity of the original immunoglobulin, despite the removal of the constant regions and the introduction of the linker. The scFv is often high-yield and low-cost produced in bacteria cell cultures such as E. coli (Bassi et al 2000). Besides, the his-tag and other markers can be added to the fusion protein by genetic engineering.…”

Section: Characterization Of the Mixed Sam For Antibody Immobilizationmentioning

confidence: 99%

A chelating-bond breaking and re-linking technique for rapid re-immobilization of immune micro-sensors

et al. 2011

Biomed Microdevices

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With high sensitivity and specificity to antigen, immune micro-sensors can be used in rapid detection of pathogenic microbial. This study proposes and develops a method for rapidly regeneration of antibody on a resonant micro-cantilever sensor. A nitrilotriacetic acid (NTA) derivative is synthesized with cystine and bromoacetic acid, then added with 2-mercaptoethanol to prepare a mixed self-assembled monolayer (SAM) on Au (111) surface of the cantilever. Ni²⁺ ions are thereafter chelated on the mixed SAM to form a breakable and re-linkable chelating-bond layer. Repeatable cycles of antibody immobilization and erasing are experimentally validated with a detectable marker of synthesized biotinylated poly peptides harboring six histidine residues (named as His-Bio). Two distinguished pathogenic microbial, Escherichia. coli O157:H7 and Bacillus Anthracis, are detected with the rapidly regenerated sensor. The E. coli O157:H7 sensor exhibits a three-time repeated detection to the 10³ CFU/ml concentration microbial. Then, an E. coli O157:H7 sensor is eluted with Tris-HCl (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH = 3.0) and rapidly reconstructed into a B. Anthracis sensor by changing the re-immobilized antibody. The cantilever sensor no longer responses to E. coli O157:H7 even in a high concentration of 10⁷ CFU/ml. In contrast, the sensor is experimentally confirmed being resoluble to low concentration B. Anthracis at 10³ spores/ml level. The proposed fast regeneration method is promising in repeatedly or multi-target detection applications of micro/nano immune-sensors, e.g. the resonant micro-cantilevers.

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“…In particular, the repeating domain in the central region of INP is not essential for the protein to anchor on the outer membrane, so it can be reduced or deleted when used as anchoring motif. To date, proteins successfully expressed in E. coli and displayed on the outer membrane using the INP fusion system include various bacterial enzymes (Jung et al, 1998a(Jung et al, , 1998b(Jung et al, , 2003Yim et al, 2006), viral epitopes (Kang et al, 2003) and several eukaryotic proteins including single chain antibody (Kwak et al, 1999;Bassi et al, 2000).…”

Section: Al 2006)mentioning

confidence: 99%

Stable expression and secretion of polyhydroxybutyrate depolymerase of Paucimonas lemoignei in Escherichia coli

et al. 2008

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An efficient strategy for the expression and secretion of extracellular polyhydroxybutyrate depolymerase (PhaZ1) of Paucimonas lemoignei in Escherichia coli was developed by employing the signal peptide of PhaZ1 and a truncated ice nucleation protein anchoring motif (INPNC). Directly synthesized mature form of Phaz1 was present in the cytoplasm of host cells as inclusion bodies, while a construct containing Phaz1 and its own N-terminal signal peptide (PrePhaz1) enabled the secretion of active Phaz1 into the extracellular medium. However, the PrePhaz1 construct was harmful to the host cell and resulted in atypical growth and instability of the plasmid during the cultivation. In contrast, INPNC-Phaz1 and INPNC-PrePhaz1 fusion constructs did not affect growth of host cells. INPNC-Phaz1 was successfully displayed on the cell surface with its fusion form, but did not retain Phaz1 activity. In the case of INPNC-PrePhaz1, the initially synthesized fusion form was separated by precise cleavage of the signal peptide, and active Phaz1 was consequently released into the culture medium. The amount of Phaz1 derived from E. coli (INPNC-PrePhaz1) was almost twice as great as that directly expressed from E. coli (PrePhaz1), and was predominantly (approximately 85%) located in the periplasm when cultivated at 22 degrees C but was efficiently secreted into the extracellular medium when cultivated at 37 degrees C.

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Expression of Single Chain Antibodies (ScFvs) for c‐myc Oncoprotein in Recombinant Escherichiacoli Membranes by Using the Ice‐Nucleation Protein of Pseudomonassyringae

Cited by 17 publications

References 33 publications

Bioremediation of Organophosphorus Pesticides by Surface-Expressed Carboxylesterase from Mosquito on Escherichia Coli

Bioremediation of Organophosphorus Pesticides by Surface-Expressed Carboxylesterase from Mosquito on Escherichia Coli

A chelating-bond breaking and re-linking technique for rapid re-immobilization of immune micro-sensors

Stable expression and secretion of polyhydroxybutyrate depolymerase of Paucimonas lemoignei in Escherichia coli

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