Efficient construct of a large and functional scFv yeast display library derived from the ascites B cells of ovarian cancer patients by three-fragment transformation-associated recombination

Applied Microbiology and Biotechnology
Xiaopeng YuanAizhi Zhao

Abstract

Over the past decade, yeast display technology has emerged as a powerful tool for the isolation of high-affinity immunoglobulin fragments with potential utility as clinical diagnostic and therapeutic reagents. Despite significant refinement of the various methodologies underpinning library construction and selections, certain aspects remain challenging and process limiting. We have sought to significantly improve the robustness of the single-chain Fv (scFv) library construction step by overcoming the technical inefficiencies frequently encountered during the PCR-mediated assembly of scFvs from the discrete heavy and light V-domain repertoires. Using a novel primer set designed to provide maximum amplification coverage of the known germ-line V-domain repertoire, we have exploited the potential of the in vivo homologous gap-repair apparatus of Saccharomyces cerevisiae to assemble intact scFvs directly from co-transformed PBMC-derived VH, VL, and linearized vector component fragments. We have successfully applied this three-fragment assembly strategy to construct a large (>10(9)) scFv yeast display library from the ascites immune repertoire of ovarian cancer patients and validated the approach by applying FACS-based sorting to rea...Continue Reading

References

Dec 5, 1991·Journal of Molecular Biology·J D MarksG Winter
Jun 1, 1997·Nature Biotechnology·E T Boder, K D Wittrup
Apr 8, 1998·Immunotechnology : an International Journal of Immunological Engineering·D Sblattero, A Bradbury
May 30, 1998·Proceedings of the National Academy of Sciences of the United States of America·M D SheetsE Lindqvist
Oct 2, 1998·Cancer Immunology, Immunotherapy : CII·T IizasaH Ohwada
Dec 11, 1999·Nucleic Acids Research·G Johnson, T T Wu
Jun 21, 2001·The Biochemical Journal·Y WenM G O'Rand
Feb 24, 2004·Nucleic Acids Research·Jeffrey S SwersK Dane Wittrup
Apr 20, 2004·FEBS Letters·Jane M Weaver-FeldhausMichael J Feldhaus
Dec 21, 2004·Nucleic Acids Research·Ida RetterWerner Müller
Aug 6, 2005·The Journal of Immunology : Official Journal of the American Association of Immunologists·Beatrix KotlanMark C Glassy
Jun 17, 2006·Biochemical and Biophysical Research Communications·Hae-Won LeeYong-Sung Kim
Oct 19, 2006·Experimental and Molecular Pathology·Saumya PandeyDevendra K Agrawal
Nov 23, 2006·Journal of Immunological Methods·Nathalie SchollerNicole Urban
Jan 12, 2008·The American Journal of Pathology·Sven ChristianHellmut G Augustin
Dec 17, 2008·Proceedings of the National Academy of Sciences of the United States of America·Daniel G GibsonClyde A Hutchison
Apr 21, 2009·Journal of Molecular Biology·Laura M WalkerDennis R Burton
Feb 5, 2010·Protein Engineering, Design & Selection : PEDS·Lorenzo BenatuilChung-Ming Hsieh
May 25, 2010·The Journal of Immunology : Official Journal of the American Association of Immunologists·Scott D BoydAndrew M Collins

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Citations

Aug 9, 2018·Microbial Cell Factories·Hongting TangXiaoming Bao

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