Combinatorial libraries offer an attractive approach towards exploring protein sequence, structure and function. Although several strategies introduce sequence diversity, the likelihood of identifying proteins with novel functions is increased when the library of genes encodes for folded and soluble structures. Here we present the first application of the binary patterning approach of combinatorial protein library design to the unique central linker region of the highly-conserved protein, calmodulin (CaM). We show that this high-quality approach translates very well to the CaM protein scaffold: All library members over-express and are functionally diverse, having a range of conformations in the presence and absence of calcium as determined by circular dichroism spectroscopy. Collectively, these data support that the binary patterning approach, when applied to the highly-conserved protein fold, can yield large collections of folded, soluble and highly-expressible proteins.
|Number of pages||6|
|Journal||Protein Expression and Purification|
|State||Published - Feb 2011|
Bibliographical noteFunding Information:
The authors thank Dr. Tom Vanaman for providing the pETCaMI expression plasmid and discussions. The authors also thank Kristen Kelps, Catherine Werner, Victoria Dunlap, Aparna Vital, and Sudipa Chowdhury for technical assistance. All spectroscopy was performed at the University of Kentucky Center of Structural Biology. All sequencing was performed at the University of Kentucky Advanced Genetics Technology Center. M.L.B. conducted research as part of his University of Kentucky Undergraduate Honors Thesis project. This study was supported by funds from University of Kentucky Office of Undergraduate Research and Creative Activities/eUreKa (M.L.B.) , NIH National Center for Research Resources (NCRR) grant P20 RR020171 (L.H.B.), the Kentucky Science and Engineering Foundation (Grant Agreement# KSEF-148-502-07-201 with the Kentucky Science and Technology Corporation) (L.H.B. ), and the University of Kentucky College of Medicine start-up funds (L.H.B.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
- Binary patterning
- Combinatorial library
- Protein design
- Protein engineering
ASJC Scopus subject areas