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Protein Science (2004), 13:1851-1858. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Monoclonal antibodies assisting refolding of firefly luciferase

Qin Xu1,3, Zhiqun Xie1, Jianfang Ding1, Sheng-Xiang Lin2 and Genjun Xu1

1 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
2 Laboratory of Oncology and Molecular Endocrinology, Laval University Medical Center, Québec G1V 4G2, Canada

(RECEIVED February 13, 2004; FINAL REVISION April 7, 2004; ACCEPTED April 8, 2004)



Abstract

The reactivation efficiency in the refolding of denatured luciferase in the presence and the absence of monoclonal antibodies (mAbs) has been studied. Luciferase could be partially reactivated when the protein was denatured in high concentrations of guanidium chloride (GdmCl; >4.5 M) and the refolding was carried out in very low protein concentrations. The refolding yield was, however, significantly lower when it was performed on luciferase that had been denatured with lower concentrations of GdmCl. The efficiency of refolding decreases when the formation of aggregates increases. Three of the five luciferase mAbs tested (4G3, N2E3, S2G10) dramatically increased the yield of reactivation and simultaneously eliminated the formation of aggregates. It is proposed that these mAbs assisted the refolding of luciferase by binding to the exposed hydrophobic surface of the refolding intermediate, thus preventing it from aggregating. The epitopes interacting with these refolding-assisting mAbs are all located in the A-subdomain of the N-terminal region of luciferase. These results have also shed light on the structural features of the intermediate and its interface involved in protein aggregate formation, contributing to the understanding of the protein folding mechanism.

Keywords: luciferase; protein unfolding and refolding; protein aggregation; monoclonal antibodies; epitopes; enzyme activity

Abbreviations: ANS, 1-anilinonaphthalene-8-sulphonic acid • BSA, bovine serum albumin • CD, circular dichroism • Cm, midpoint • GdmCl, guanidinium chloride • mAb, monoclonal antibody

Reprint requests to: GenJun Xu, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-yang Road, Shanghai 200031, China; e-mail: gjxu{at}sibs.ac.cn; fax: 86-21-54921257.

3 Present address: Gladstone Institute of Neurological Disease and Glad-stone Institute of Cardiovascular Disease, University of California, San Francisco, CA 94141-9100, USA.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04699904.


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