High-throughput assays are indispensable for comprehensive functional proteome research. The development of these techniques has been promoted again by the successful completion of complete genomes, including the human. Of great importance for achieving this goal is the development of new protein capture tools for the detection and identification of specific proteins. These new set of capture reagents should be stable to thermal and proteolytic degradation, have high affinity, easy to produce and present low cross-reactivity. In response to this challenge, we propose the use of cell-based libraries of cyclotides for selecting specific cyclotide sequences against particular protein targets. Cyclotides are a new emerging family of plant-derived backbone-cyclized polypeptides (~28-37 amino acids long) that share a disulfidestabilized core (3 disulfide bonds) characterized by an unusual knotted structure. Their unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to thermal and enzymatic degradation. Cyclotides have been associated with a range of biological functions such as uterotonic activity, inhibition of trypsin and neurotensin binding, cytotoxicity, anti-HIV, antimicrobial, and insecticidal activity. Together, these characteristics make cyclotides ideal candidates to be used as molecular scaffolds for the discovery of stable high affinity ligands against particular biomolecular targets thus replacing the less stable antibody-based scaffold, which have been traditionally used as the protein capture reagent of choice. We have recently demonstrated the intriguing possibility of generating libraries of cyclotides inside living bacterial cells. Biologically-generated libraries can be screened inside the cell using cell-based reporters (both based on FRET or lethality based) for the selection of particular cyclotide sequences able to bind a particular protein target using high throughput methods such as fluorescence-activated cell Sorting (FACS). Selected cyclotide sequences can be then immobilized using a micro-array format onto appropriate solid supports. Of particular interest is the use of protein trans-splicing for the immobilization of these micro-proteins onto solid supports. This approach allows the site-specific and traceless immobilization of particular proteins/polypeptides (for example linearized cyclotides) from mixtures without any need to purify or re-concentrate the ligand to be immobilized. More importantly, this approach can be interfaced with cell-free expression systems for the rapid and high throughput of specific cyclotide based microarrays.

Public Health Relevance

: This proposal presents an innovative approach for the screening and selection on a new type of extremely stable protein capture reagents in combination with a new versatile way of ligand immobilization for the rapid production of cyclotide-based micro-arrays. We anticipate that these technologies will have a large impact in science in general but will also provide the diagnostic and prognostic benefits of clinical proteomics to individual patients and outpatient clinics around the world.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM090323-05
Application #
8528623
Study Section
Special Emphasis Panel (ZRG1-BCMB-A (51))
Program Officer
Fabian, Miles
Project Start
2009-09-30
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
5
Fiscal Year
2013
Total Cost
$385,033
Indirect Cost
$147,359
Name
University of Southern California
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
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Jagadish, Krishnappa; Borra, Radhika; Lacey, Vanessa et al. (2013) Expression of fluorescent cyclotides using protein trans-splicing for easy monitoring of cyclotide-protein interactions. Angew Chem Int Ed Engl 52:3126-31
Ji, Yanbin; Majumder, Subhabrata; Millard, Melissa et al. (2013) In vivo activation of the p53 tumor suppressor pathway by an engineered cyclotide. J Am Chem Soc 135:11623-33
Borra, Radhika; Camarero, Julio A (2013) Recombinant expression of backbone-cyclized polypeptides. Biopolymers 100:502-9
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Gould, Andrew; Li, Yilong; Majumder, Subhabrata et al. (2012) Recombinant production of rhesus ?-defensin-1 (RTD-1) using a bacterial expression system. Mol Biosyst 8:1359-65
Borra, Radhika; Dong, Dezheng; Elnagar, Ahmed Y et al. (2012) In-cell fluorescence activation and labeling of proteins mediated by FRET-quenched split inteins. J Am Chem Soc 134:6344-53
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Contreras, Janette; Elnagar, Ahmed Y O; Hamm-Alvarez, Sarah F et al. (2011) Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways. J Control Release 155:134-43

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