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Phage surface presentation and secretion of antibody fragments using an adaptable phagemid vector

Phage surface presentation and secretion of antibody fragments using an adaptable phagemid vector Phagemid vectors have been developed which promise to supersede hybridoma technology for the selection and production of human antibodies. We have modified an existing phagemid vector to improve the stability of synthesized soluble antibody fragments. The vector allows the antibody fragment to be produced: i) as a soluble protein incorporating a stable carboxyl terminal octapeptide (FLAG) or, ii) on the surface of a bacteriophage fused to a minor coat protein (the gene III protein). The antibody gene encoding the well characterized monoclonal antibody NC10 (an antibody that recognizes the neuraminidase of the influenza strain N9) was inserted as a single chain Fν construct into the phagemid vectors pHFA and pHFA/SacII. Western blotting, ELISA and electron microscopy studies showed that recombinant clones could be manipulated to either synthesize soluble protein into the peri plasm or present the protein on the surface of bacteriophage. Cosynthesis of GroEL and GroES chaperon ins resulted in complete proteolysis of the scFνNC10-FLAG-gIIIp fusion product and did not improve total phage production. Coexpression of chaperonins should be used with caution for library construction due to the expected selection pressure for protease resistant gene III fusions. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Antibodies IOS Press

Phage surface presentation and secretion of antibody fragments using an adaptable phagemid vector

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Publisher
IOS Press
Copyright
Copyright © 1994 by IOS Press, Inc
ISSN
1093-2607
eISSN
1875-869X
DOI
10.3233/HAB-1994-51-207
Publisher site
See Article on Publisher Site

Abstract

Phagemid vectors have been developed which promise to supersede hybridoma technology for the selection and production of human antibodies. We have modified an existing phagemid vector to improve the stability of synthesized soluble antibody fragments. The vector allows the antibody fragment to be produced: i) as a soluble protein incorporating a stable carboxyl terminal octapeptide (FLAG) or, ii) on the surface of a bacteriophage fused to a minor coat protein (the gene III protein). The antibody gene encoding the well characterized monoclonal antibody NC10 (an antibody that recognizes the neuraminidase of the influenza strain N9) was inserted as a single chain Fν construct into the phagemid vectors pHFA and pHFA/SacII. Western blotting, ELISA and electron microscopy studies showed that recombinant clones could be manipulated to either synthesize soluble protein into the peri plasm or present the protein on the surface of bacteriophage. Cosynthesis of GroEL and GroES chaperon ins resulted in complete proteolysis of the scFνNC10-FLAG-gIIIp fusion product and did not improve total phage production. Coexpression of chaperonins should be used with caution for library construction due to the expected selection pressure for protease resistant gene III fusions.

Journal

Human AntibodiesIOS Press

Published: Jan 1, 1994

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