Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation

PloS One
Aerin YoonJunho Chung

Abstract

For the site-directed conjugation of chemicals and radioisotopes to the chicken-derived single-chain variable fragment (scFv), we investigated amino acid residues replaceable with cysteine. By replacing each amino acid of the 157 chicken variable region framework residues (FR, 82 residues on VH and 75 on VL) with cysteine, 157 artificial cysteine mutants were generated and characterized. At least 27 residues on VL and 37 on VH could be replaced with cysteine while retaining the binding activity of the original scFv. We prepared three VL (L5, L6 and L7) and two VH (H13 and H16) mutants as scFv-Ckappa fusion proteins and showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all five mutants. Because the charge around the cysteine residue affects the in vivo stability of thiol-maleimide conjugation, we prepared 16 charge-variant artificial cysteine mutants by replacing the flanking residues of H13 with charged amino acids and determined that the binding activity was not affected in any of the mutants except one. We prepared four charge-variant H13 artificial cysteine mutants (RCK, DCE, ECD and ECE) as scFv-Ckappa fusion proteins and confirmed that the reactivity of the sulfhydryl group on...Continue Reading

References

Aug 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·J S HustonR Crea
Oct 21, 1988·Science·R E BirdM Whitlow
Apr 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·J D RodwellT J McKearn
Aug 1, 1995·Proceedings of the National Academy of Sciences of the United States of America·O BoussifJ P Behr
Jul 6, 2000·The Journal of Biological Chemistry·J B StimmelF C Kull
Mar 26, 2004·Nature Reviews. Immunology·Jenny M Woof, Dennis R Burton
Sep 10, 2005·Nature Biotechnology·Philipp Holliger, Peter J Hudson
Sep 10, 2005·Nature Biotechnology·Anna M Wu, Peter D Senter
Sep 10, 2005·Nature Biotechnology·Gregory P Adams, Louis M Weiner
Sep 22, 2005·Bioconjugate Chemistry·Michael M C SunStephen C Alley
Nov 16, 2005·Annual Review of Genetics·Nancy Maizels
Jul 22, 2008·Nature Biotechnology·Jagath R JunutulaWilliam Mallet
Jan 23, 2010·Bioconjugate Chemistry·Vladimir VoynovBernhardt L Trout
Feb 25, 2010·Molecular Therapy : the Journal of the American Society of Gene Therapy·Richard A MorganSteven A Rosenberg
Dec 2, 2011·The Journal of Immunology : Official Journal of the American Association of Immunologists·Leeying WuWilliam J J Finlay
Apr 5, 2012·Clinical & Developmental Immunology·Zuhaida Asra AhmadMuhajir Hamid
Nov 14, 2012·The Journal of Biological Chemistry·Heather H ShihWilliam J J Finlay
Jul 23, 2013·Frontiers in Oncology·Caroline Bodet-MilinFrançoise Kraeber-Bodéré
Nov 28, 2013·Annual Review of Medicine·David M BarrettCarl H June
Jul 23, 2014·Molecular Imaging and Biology : MIB : the Official Publication of the Academy of Molecular Imaging·Masashi UedaHideo Saji
Sep 23, 2014·Experimental & Molecular Medicine·Yujean LeeJunho Chung
Dec 17, 2014·Immunological Reviews·Saar Gill, Carl H June
Mar 10, 2015·Nature Biotechnology·Cormac Sheridan

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Datasets Mentioned

BETA
KP764766

Methods Mentioned

BETA
fluorescence microscopy
amidation
PCR
electrophoresis

Software Mentioned

SigmaPlot
PyMOL

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