Nature Biotechnology
20, 1246 - 1250 (2002)
Published online: 11 November 2002; | doi:10.1038/nbt757
Growth factor engineering by degenerate homoduplex gene family recombinationWayne M. Coco1, 2, Lance P. Encell1, 2, William E. Levinson1, Michael J. Crist1, 3, A. Katrina Loomis1, 2, Laura L. Licato1, 4, Joseph J. Arensdorf1, 5, Nicole Sica1, 2, Philip T. Pienkos1, 6
& Daniel J. Monticello1, 71
Enchira Biotechnology Corporation, 4200 Research Forest Drive, The Woodlands, TX 77381. 2
Current address: Tanox, Inc., Houston, TX 77025. 3
Current address: Lexicon Genetics, Inc., The Woodlands, TX 77381. 4
Current address: Isolagen, Inc., Houston, TX 77042. 5
Current address: Baker Petrolite, Sugar Land, TX 77478. 6
Current address: Fermentation Developments, The Woodlands, TX 77381. 7
Current address: Molecular Logix Inc., 4200 Research Forest Drive, The Woodlands, TX 77381.
Correspondence should be addressed to Wayne M. Coco wcoco@tanox.comThere is great interest in engineering human growth factors as potential therapeutic agonists and antagonists. We approached this goal with a synthetic DNA recombination method. We aligned a pool of "top-strand" oligonucleotides incorporating polymorphisms from mammalian genes encoding epidermal growth factor (EGF) using multiple polymorphic "scaffold" oligonucleotides. Top strands were then linked by gap filling and ligation. This approach avoided heteroduplex annealing in the linkage of highly degenerate oligonucleotides and thus achieved completely random recombination. Cloned genes from a human−mouse chimeric library captured every possible permutation of the parental polymorphisms, creating an apparently complete recombined gene-family library, which has not been previously described. This library yielded a chimeric protein whose agonist activity was enhanced 123-fold. A second library from five mammalian EGF homologs possessed the highest reported recombination density (1 crossover per 12.4 bp). The five-homolog library yielded the strongest-binding hEGF variant yet reported. In addition, it contained strongly binding EGF variants with antagonist properties. Our less biased approach to DNA shuffling should be useful for the engineering of a wide variety of proteins.
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