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PEDS Advance Access published online on November 24, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzn071
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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

M. Arbabi-Ghahroudi1, R. To1, N. Gaudette1,4, T. Hirama1, W. Ding1, R. MacKenzie1,2 and J. Tanha1,2,3,5

1Institute for Biological Sciences, National Research Council of Canada, Ottawa, ON, Canada K1A 0R6 2Department of Environmental Biology, Ontario Agricultural College, University of Guelph, Guelph, ON, Canada N1G 2W1 3Depeartment of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada K1 N 6N5

5To whom correspondence should be addressed. E-mail: jamshid.tanha{at}nrc-cnrc.gc.ca

When panned with a transient heat denaturation approach against target enzymes, a human VH (antibody heavy chain variable domain) phage display library yielded VHs with composite characteristics of binding, non-aggregation and reversible thermal unfolding. Moreover, selection was characterized by enrichment for VHs 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 VHs as inferred here by a pI distribution analysis, reported here, of pools of human and camelid VH and VHH (camelid heavy chain antibody VH) 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 VH domains/libraries for non-aggregation optimization. In addition, calculation of theoretical pIs of VHs selected by panning may be used for rapid and precise identification of non-aggregating VHs.

Keywords: disulfide-bonded loops/isoelectric point/non-aggregating VH/phage display library and panning/single-domain antibody

Received August 7, 2008; revised October 28, 2008; accepted October 31, 2008.

* This is National Research Council of Canada Publication 42530.

4 Present address: Canadian Border Services Agency, Ottawa, ON, Canada K2E 6T7.


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