PEDS Advance Access originally published online on March 30, 2006
Protein Engineering Design and Selection 2006 19(6):245-253; doi:10.1093/protein/gzl006
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Improved mutants from directed evolution are biased to orthologous substitutions
1 Division of Biological Engineering, Massachusetts Institute of Technology Cambridge, MA 02139, USA 2 Department of Chemical Engineering, Massachusetts Institute of Technology Cambridge, MA 02139, USA 3Present address: Department of Bioengineering, Stanford University Stanford, CA 94305, USA 4Present address: Department of Molecular Science and Technology, Ajou University Korea
5To whom correspondence should be addressed. E-mail: wittrup{at}mit.edu
We have engineered human epidermal growth factor (EGF) by directed evolution through yeast surface display for significantly enhanced affinity for the EGF receptor (EGFR). Statistical analysis of improved EGF mutants isolated from randomly mutated yeast-displayed libraries indicates that mutations are biased towards substitutions at positions exhibiting significant phylogenetic variation. In particular, mutations in high-affinity EGF mutants are statistically biased towards residues found in orthologous EGF species. This same trend was also observed with other proteins engineered through directed evolution in our laboratory (EGFR, interleukin-2) and in a meta-analysis of reported results for engineered subtilisin. By contrast, reported loss-of-function mutations in EGF were biased towards highly conserved positions. Based on these findings, orthologous mutations were introduced into a yeast-displayed EGF library by a process we term shotgun ortholog scanning mutagenesis (SOSM). EGF mutants with a high frequency of the introduced ortholog mutations were isolated through screening the library for enhanced binding affinity to soluble EGFR ectodomain. These mutants possess a 30-fold increase in binding affinity over wild-type EGF to EGFR-transfected fibroblasts and are among the highest affinity EGF proteins to be engineered to date. Collectively, our findings highlight a general approach for harnessing information present in phylogenetic variability to create useful genetic diversity for directed evolution. Our SOSM method exploits the benefits of library diversity obtained through complementary methods of error-prone PCR and DNA shuffling, while circumventing the need for acquisition of multiple genes for family or synthetic shuffling.
Keywords: epidermal growth factor/epidermal growth factor receptor/in vitro evolution/protein engineering/yeast display
Received October 21, 2005; revised January 10, 2006; accepted February 16, 2006.
Edited by Andrew Bradbury
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Paramesvaran, E. G. Hibbert, A. J. Russell, and P. A. Dalby Distributions of enzyme residues yielding mutants with improved substrate specificities from two different directed evolution strategies Protein Eng. Des. Sel., July 1, 2009; 22(7): 401 - 411. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Nolan and L. Yang The flow of cytometry into systems biology Brief Funct Genomic Proteomic, July 4, 2007; (2007) elm011v1. [Abstract] [Full Text] [PDF]
|
|