Growth factor engineering by degenerate homoduplex gene family recombination.
There 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.
Coco
Wayne M
WM
Enchira Biotechnology Corporation, 4200 Research Forest Drive, The Woodlands, TX 77381, USA. wcoco@tanox.com
Encell
Lance P
LP
Levinson
William E
WE
Crist
Michael J
MJ
Loomis
A Katrina
AK
Licato
Laura L
LL
Arensdorf
Joseph J
JJ
Sica
Nicole
N
Pienkos
Philip T
PT
Monticello
Daniel J
DJ
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004815:Epidermal Growth Factor / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D006736:Horses; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D051381:Rats; D011994:Recombinant Proteins / Q000145:classification / Q000235:genetics; D011995:Recombination, Genetic; D016415:Sequence Alignment / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods; D013552:Swine
D019151:Peptide Library; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor