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.
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