Protein Engineering, Vol 11, 1267-1276, Copyright © 1998 by Oxford University Press
HJ de Haard, B Kazemier, A van der Bent, P Oudshoorn, P Boender, B van Gemen, JW Arends and HR Hoogenboom
While studying the expression of single-chain antibodies (scFv) derived
from several murine monoclonal antibodies, we found that residue 6 in
Framework region 1 of the heavy chain variable domain plays a crucial role
in antibody folding. Binding activity of three murine antibodies with a
heavy chain variable region (VH) subgroup IIA was completely lost when at
this position the wild-type residue glutamine (Q) was substituted by
glutamate (E). Increased sensitivity towards trypsin digestion of soluble
scFv suggested that the lack of binding activity was caused by incorrect
folding of Q6E mutants. Grafting of the three additional class IA derived
FR1 residues, based upon the comparison between both classes of VH
sequences, on to the 'defect' subgroup IIA sequence, partially restored the
antigen binding activity of the Q6E- containing scFv. Our results suggest
that residue 6 of the heavy chain may be part of a folding nucleus,
involving the first two beta-strands of Framework region 1. The
evolutionary conservation of either glutamine or glutamate at position 6 in
different antibody families may well indicate that within immunoglobulin VH
domains, different family specific folding nuclei have evolved.
ARTICLES
Absolute conservation of residue 6 of immunoglobulin heavy chain variable regions of class IIA is required for correct folding
Biosciences Research Unit, Organon Teknika, Boxtel, The Netherlands. hans-de.haard@unilever.com
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