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 Fv 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 periplasm or present the protein on the surface of bacteriophage. Cosynthesis of GroEL and GroES chaperonins resulted in complete proteolysis of the scFvNC10-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.
CSIRO Division of Biomolecular Engineering, Parkville Vic., Australia.
M
M
Lah
A
A
Goldstraw
J F
JF
White
O
O
Dolezal
R
R
Malby
P J
PJ
Hudson
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics*; D000915:Antibody Affinity; D001483:Base Sequence; D018833:Chaperonins / Q000096:biosynthesis / Q000235:genetics; D003090:Coliphages / Q000235:genetics* / Q000276:immunology* / Q000648:ultrastructure; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering; D005822:Genetic Vectors; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D004247:DNA; D018833:Chaperonins