NDLI: Optimized expression and specific activity of IL-12 by directed molecular evolution.

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Optimized expression and specific activity of IL-12 by directed molecular evolution.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Stemmer, Willem P. C. Leong, Steven R. Chang, Jean C. C. Ong, Randal Dawes, Glenn Punnonen, Juha
Description	Author Affiliation: Leong SR ( Department of Vaccines, Maxygen, Incorporated, 515 Galveston Drive, Redwood City, CA 94063, USA.);
Abstract	DNA delivery of IL-12 has shown promise in reducing the toxic side effects associated with administration of recombinant human (h)IL-12 protein while maintaining the ability to inhibit tumor growth and abolish tumor metastases in animal models. We have developed a more potent version of IL-12 by using DNA shuffling and screening to improve its expression in human cells and specific activity on human T cells. The most improved evolved IL-12 (EvIL-12) derived from seven mammalian genes encoding both the p35 and p40 subunits of IL-12 showed a 128-fold improvement in human T cell proliferation compared with native hIL-12 during the initial screening of supernatants from transected cells. When purified hIL-12 and EvIL-12 proteins were compared in vitro in human T cell proliferation and Th1 differentiation assays, it was demonstrated that EvIL-12 exhibited a concomitant 10-fold increase in the specific activity of the protein compared with hIL-12. Furthermore, DNA shuffling improved the level of expression and homogeneity of the heterodimer synthesized by 293 human embryonic kidney cells transfected with EvIL-12 by at least 10-fold. Molecular analysis of the variant revealed strategic placement of amino acid substitutions that potentially may facilitate heterodimer formation and product expression. The enhanced expression and biological activity of EvIL-12 may improve the effectiveness of IL-12 gene-based vaccines and therapeutics without the toxic side effects sometimes associated with hIL-12 protein administration.
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	3
Volume Number	100
Language	English
Publisher	National Academy of Sciences
Publisher Date	2003-02-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	DNA Genetic Techniques Interleukin-12 Biosynthesis Genetics Amino Acid Sequence Animals COS Cells Cell Differentiation Cell Division Cell Line Cells, Cultured Dimerization Directed Molecular Evolution Dose-Response Relationship, Drug Gene Library Glycosylation Models, Molecular Molecular Sequence Data T-Lymphocytes Th1 Cells Transfection Research Support, U.S. Gov't, Non-P.H.S. Multidisciplinary
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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