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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Roberts, Richard W. Olson, C. Anders |
| Description | Country affiliation: United States Author Affiliation: Olson CA ( Biochemistry and Molecular Biophysics Option, California Institute of Technology, Pasadena, California 91125, USA.) |
| Abstract | Protein libraries based on natural scaffolds enable the generation of novel molecular tools and potential therapeutics by directed evolution. Here, we report the design and construction of a high complexity library (30 x 10(13) sequences) based on the 10th fibronectin type III domain of human fibronectin (10FnIII). We examined the bacterial expression characteristics and stability of this library using a green fluorescent protein (GFP)-reporter screen, SDS-PAGE analysis, and chemical denaturation, respectively. The high throughput GFP reporter screen demonstrates that a large fraction of our library expresses significant levels of soluble protein in bacteria. However, SDS-PAGE analysis of expression cultures indicates the ratio of soluble to insoluble protein expressed varies greatly for randomly chosen library members. We also tested the stabilities of several representative variants by guanidinium chloride denaturation. All variants tested displayed cooperative unfolding transitions similar to wild-type, and two exhibited free energies of unfolding equal to wild-type 10FnIII. This work demonstrates the utility of GFP-based screening as a tool for analysis of high-complexity protein libraries. Our results indicate that a vast amount of protein sequence space surrounding the 10FnIII scaffold is accessible for the generation of novel functions by directed as well as natural evolution. |
| ISSN | 09618368 |
| e-ISSN | 1469896X |
| Journal | Protein Science |
| Issue Number | 3 |
| Volume Number | 16 |
| Language | English |
| Publisher | Wiley-Blackwell (on behalf of The Protein Society) |
| Publisher Date | 2007-03-01 |
| Publisher Place | United States |
| Access Restriction | Open |
| Subject Keyword | Fibronectins Chemistry Protein Engineering Combinatorial Chemistry Techniques Electrophoresis, Polyacrylamide Gel Escherichia Coli Genetics Metabolism Biosynthesis Genes, Reporter Green Fluorescent Proteins Guanidine Oligonucleotides Protein Denaturation Protein Folding Solubility Research Support, N.i.h., Extramural Research Support, Non-u.s. Gov't Research Support, U.s. Gov't, Non-p.h.s. Discipline Biochemistry |
| Content Type | Text |
| Resource Type | Article |
| Subject | Medicine Molecular Biology Biochemistry |
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