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Chaperonin Assisted Phage Display of Antibody Fragments on Filamentous Bacteriophages

Bio/Technology volume 11, pages 503–507 (1993)Cite this article

Abstract

We have used the GroE chaperonins to assist in the packing of a new phage display vector, pEXmide3. Titers of the packed phagemid increased almost 200-fold from ∼ 4×1011 cfu/ml, without coexpression of the GroE proteins, to ∼7×l013 cfu/ml with their coexpression. Equal titers of non-assisted and assisted phagestocks exhibited the same antigen specificity and ELISA reactivity, indicating the same frequency of displayed Fab-fragments. While the diversity of antibody libraries depends on the bacterial transformation efficiency, the copy number of each antibody is determined by subsequent amplification of the phage, thus chaperonin assisted phagemid packing in bacteriophage M13 can be used as a general and simple tool to increase the amplification level of expressed Fab fragments. pEXmide3 was developed for display of Fab and single chain Fv-fragments (scFv), using restriction enzymes that do not cut, or cut with low frequencies, in genes encoding immunoglobulin variable domains. The vector allows cloning of genes for the variable domains Unking these to predetermined human constant domains or cloning of the entire light and heavy Fab chains. A modification of the pelB leader sequence, with a glutamine to alanine substitution at residue 18, was used for export of the light chain.

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Author notes

  1. Carl A. K. Borrebaeck: Corresponding author.

Authors and Affiliations

  1. Department of Immunotechnology, Lund University, P.O. Box 7031, Lund, S-220 07, Sweden

    Eskil Söderfind, Ann Catrin Simonsson Lagerkvist, Marta Dueñas, Ann-Christin Malmborg, Lena Danielsson & Carl A. K. Borrebaeck

  2. Division of Immunotechnology and Diagnostics, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana, 10600, Cuba

    Marta Ayala

Authors
  1. Eskil Söderfind
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  2. Ann Catrin Simonsson Lagerkvist
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  3. Marta Dueñas
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  4. Ann-Christin Malmborg
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  5. Marta Ayala
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  7. Carl A. K. Borrebaeck
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Söderfind, E., Lagerkvist, A., Dueñas, M. et al. Chaperonin Assisted Phage Display of Antibody Fragments on Filamentous Bacteriophages. Nat Biotechnol 11, 503–507 (1993). https://doi.org/10.1038/nbt0493-503

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