Traditional protein site-directed mutagenesis has served biochemists for many years but is lacking when one seeks to construct libraries of mutants such as in alanine scanning. In protein engineering experiments, protein libraries are redundant mixtures requiring more screening or selection than would be necessary from a "perfect" mixture. This proposal seeks to create a simple, fast, and effective way to perform scanning codon mutagenesis throughout any protein coding sequence. Coupled with a reading frame selection, this will allow the rapid production of collections of proteins that contain substitutions of a defined set of amino acids, including those that contain unnatural functional groups. The proposed studies will i)develop a Mu transposon-based method for the random insertion and deletion of in-frame codon mutations from an open reading frame, ii) verify this methodology by performing whole-gene alanine scanning on an a counterselectable enzyme expressed in E. coli iii) generate a series of "smart" 2-lactamase enzyme libraries which are rich in disulfide bonds or charged amino acids and screen those libraries for improved thermostablity, and iv)use this technology to singly incorporate photo-crosslinking amino acids at every possible position throughout a multi-protein complex allowing subsequent analysis by mass-spectroscopy. PUBLIC HEALTH RELEVENCE: This proposal will develop new protein engineering technology that provides access to improved biocatalysts and therapeutic proteins. Furthermore it will provide a tool for structure-function studies on large protein complexes and membrane proteins, both of which are difficult to study using currently available methods.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM084396-05
Application #
8339452
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Edmonds, Charles G
Project Start
2008-08-01
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2014-05-31
Support Year
5
Fiscal Year
2012
Total Cost
$198,287
Indirect Cost
$61,073
Name
Virginia Commonwealth University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
Liu, Jia; Cropp, T Ashton (2013) Rational protein sequence diversification by multi-codon scanning mutagenesis. Methods Mol Biol 978:217-28
Liu, Jia; Cropp, T Ashton (2012) Experimental methods for scanning unnatural amino acid mutagenesis. Methods Mol Biol 794:187-97
Liu, Jia; Cropp, T Ashton (2012) A method for multi-codon scanning mutagenesis of proteins based on asymmetric transposons. Protein Eng Des Sel 25:67-72
Castaneda, Carlos A; Liu, Jia; Kashyap, Tanuja R et al. (2011) Controlled enzymatic synthesis of natural-linkage, defined-length polyubiquitin chains using lysines with removable protecting groups. Chem Commun (Camb) 47:2026-8
Castaneda, Carlos; Liu, Jia; Chaturvedi, Apurva et al. (2011) Nonenzymatic assembly of natural polyubiquitin chains of any linkage composition and isotopic labeling scheme. J Am Chem Soc 133:17855-68
Liu, Jia; Castaneda, Carlos A; Wilkins, Bryan J et al. (2010) Condensed E. coli cultures for highly efficient production of proteins containing unnatural amino acids. Bioorg Med Chem Lett 20:5613-6
Daggett, Kelly A; Layer, Mark; Cropp, T Ashton (2009) A general method for scanning unnatural amino acid mutagenesis. ACS Chem Biol 4:109-13