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PEDS Advance Access published online on October 20, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm038
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Article

Analysis of IgG heavy chain to light chain ratio with mutant Encephalomyocarditis virus internal ribosome entry site

Jiandong Li1,2, Congcong Zhang1, Thomas Jostock1,3,4 and Stefan Dübel1,4,5

1 Institut für Biochemie und Biotechnologie, Abteilung Biotechnologie, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany

5 To whom correspondence should be addressed. E-mail: s.duebel{at}tu-bs.de

Immunoglobulin G (IgG) is a heterotetrameric protein assembled from two identical heavy chain (HC) and two identical light chain (LC) polypeptides. The HC and LC folding and assembly are a crucial step for IgG production. It is affected by the ratio of HC to LC expression (HC:LC). To date, the HC:LC ratio was analysed mainly by cotransfection of different amounts of two monocistronic HC and LC expression plasmids, an approach biased by different transfection efficiencies. To circumvent this problem, a series of Encephalomyocarditis virus internal ribosome entry site (EMCV IRES) variants with different translation efficiencies were created and used to mediate HC translation in bicistronic constructs. HC and LC were translated from the same mRNA, which provides a more accurate method for the evaluation of the optimal ratio of HC:LC. The results show that the IgG optimal expression levels were obtained when the IRES mediated translation efficiency of the HC was about 50% compared to the cap-dependent translation of the LC. A surprisingly sharp transition to low production was shown when the ratios were below 40%. This study provides a new method to investigate the production of heterodimeric proteins in mammalian cells and adds understanding to the mechanisms of IgG folding and assembly.

Keywords: antibody engineering/EMCV IRES/folding and assembly/IgG/Poly(C)

Received January 26, 2007; revised May 29, 2007; accepted June 26, 2007.

2 Present address: Institute for Viral Diseases Control and Prevention, Chinese Centers for Diseases Control and Prevention, 100 Ying Xin Jie, Xuan Wu Qu, Beijing 100052, China

3 Present address: Novartis Pharma, Biotechnology Development, CH-4002 Basel, Switzerland

4 T.Jostock and S.Dübel contributed equally to this work.


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