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PEDS Advance Access published online on September 13, 2006
Protein Engineering Design and Selection, 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
Received July 26, 2006
Accepted August 10, 2006

Article

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

Jamshid Tanha 1 *, Thanh-Dung Nguyen 1, Andy Ng 2, Shannon Ryan 1, Feng Ni 2, and Roger MacKenzie 1

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

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


  Abstract

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.
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