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Aggregation-resistant VHs selected by in vitro evolution tend to have disulfide-bonded loops and acidic isoelectric points

From National Research Council Canada

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Affiliation
  1. National Research Council of Canada. NRC Institute for Biological Sciences
FormatText, Article
Subjectantibody; binding; calculation; Canada; chain; distribution; disulfide; domain; enzyme; evolution; identification; In vitro; libraries; linkages; protein; region; residues; selection; sequence; solubility; target; theoretical
Abstract
When panned with a transient heat denaturation approach against target enzymes, a human V(H) (antibody heavy chain variable domain) phage display library yielded V(H)s with composite characteristics of binding, non-aggregation and reversible thermal unfolding. Moreover, selection was characterized by enrichment for V(H)s with (i) an even number of disulfide forming Cys residues in complementarity-determining region (CDR) 1 and CDR3 and (ii) acidic isoelectric points. This parallels naturally occurring camelid and shark single-domain antibodies (sdAbs) which are also characterized by (i) solubility and reversible unfolding, (ii) a high occurrence of disulfide forming Cys in their CDRs, particularly, in CDR1 and CDR3 and (iii) acidic V(H)s as inferred here by a pI distribution analysis, reported here, of pools of human and camelid V(H) and V(H)H (camelid heavy chain antibody V(H)) sequences. Our results, reinforced by previous observations by others, suggest that protein acidification may yet be another mechanism nature has devised to create functional sdAbs and that this concept along with the inclusion of inter-CDR disulfide linkages may be applied to human V(H) domains/libraries for non-aggregation optimization. In addition, calculation of theoretical pIs of V(H)s selected by panning may be used for rapid and precise identification of non-aggregating V(H)s
Publication date
In
  • Protein Eng Des Sel 22, no. 2 (2009): 59–66.
LanguageEnglish
NRC numberRBABIGHAHROUDI2009B
ARBABIGHAHROUDI2008
NPARC number9374900
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Record identifier5a460cbe-8d92-4293-84ff-523423692d7f
Record created2009-07-10
Record modified2020-04-16
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