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PEDS Advance Access originally published online on September 13, 2006
Protein Engineering Design and Selection 2006 19(11):503-509; doi:10.1093/protein/gzl037
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Improving solubility and refolding efficiency of human VHs by a novel mutational approach

Jamshid Tanha1,3, Thanh-Dung Nguyen1, Andy Ng2, Shannon Ryan1, Feng Ni2 and Roger MacKenzie1

1 Institute for Biological Sciences, National Research Council of Canada Ottawa, Ontario, Canada K1A 0R6 2 Biotechnology Research Institute, National Research Council of Canada Montréal, Québec, Canada H4P 2R2

3To whom correspondence should be addressed. E-mail: jamshid.tanha{at}nrc-cnrc.gc.ca

The antibody VH domains of camelids tend to be soluble and to resist aggregation, in contrast to human VH domains. For immunotherapy, attempts have therefore been made to improve the properties of human VHs by camelization of a small set of framework residues. Here, we have identified through sequence comparison of well-folded llama VH domains an alternative set of residues (not typically camelid) for mutation. Thus, the solubility and thermal refolding efficiency of a typical human VH, derived from the human antibody BT32/A6, were improved by introduction of two mutations in framework region (FR) 1 and 4 to generate BT32/A6.L1. Three more mutations in FR3 of BT32/A6.L1 further improved the thermal refolding efficiency while retaining solubility and cooperative melting profiles. To demonstrate practical utility, BT32/A6.L1 was used to construct a phage display library from which were isolated human VHs with good antigen binding activity and solubility. The engineered human VH domains described here may be useful for immunotherapy, due to their expected low immunogenicity, and in applications involving transient high temperatures, due to their efficient refolding after thermal denaturation.

Keywords: human VH/immunogenicity/mutation/phage display library/solubility and refolding

Received July 26, 2006; accepted August 10, 2006.


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