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Record identifier : 566363
Personal Name - Primary Intelectual Responsibility : Graziano, James
Title and statement of responsibility : A novel system for the creation and screening of de novo proteins from libraries of randomly shuffled secondary structure fragments [Thesis]
Publication, Distribution,Etc. : The Scripps Research Institute, 2006
Language of the Item : eng
Dissertation of thesis details and type of degree : Ph.D
Body granting the degree : , The Scripps Research Institute
Summary or Abstract : Existing proteomes comprise an infinitesimal fraction of the total primary sequence space possible. Evolutionary processes have refined protein sequence space represented by the proteomes of all living things through natural selection. It has been theorized that modern proteomes are the product of a small set of ancestral proteins, fragments of which have recombined and mutated to build a repertoire of hybrid proteins with different functions. In the absence of selective pressure for function can we rearrange the existing sequences to identify novel folds or functions not yet sampled through evolution? The Exon Theory of Genes proposes a mechanism by which small subunits of DNA originally encoding 15 to 20 residue polypeptides comprised a pool of exons that were shuffled to create full-length genes. We present a novel system for the synthesis of a library composed of fragments corresponding to distinct secondary structure elements randomly recombined. Preliminarily screening for expression of soluble clones is via flow cytometry of transformed cells expressing the library as fusions to EGFP. Target clones isolated by FACS screen are then characterized in comparison to native proteins using biophysical methods such as CD, NMR and thermal denaturation. Other approaches to creating libraries of novel sequences to screen for native-like protein characteristics and activity rely on binary patterning of the subunits for the combinatorial pool of subunits. The libraries created in this system are defined by the existing primary sequences of E. coli proteins of known structure. The primary sequences of individual helices, strands and loops comprise the pool of combinatorially assembled genes. Characterization of this library has identified several clones with native-like properties yet in most cases possessing no significant sequence homology to known proteins. In the single case where sequence homology does exist, data suggest that it demonstrates an example of convergent evolution in vitro . The efficacy of this system for identifying soluble proteins with native characteristics having been demonstrated, it will be refined and applied to identification of novel activity..
Topical Name Used as Subject : libraries
Information of biblio record : TL
 
 
 
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