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 cyclotidebased 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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-A (51))
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Fabian, Miles
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University of Southern California
Schools of Pharmacy
Los Angeles
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Bi, Tao; Li, Yilong; Shekhtman, Alexander et al. (2018) In-cell production of a genetically-encoded library based on the ?-defensin RTD-1 using a bacterial expression system. Bioorg Med Chem 26:1212-1219
Jagadish, Krishnappa; Camarero, Julio A (2017) Recombinant Expression of Cyclotides Using Split Inteins. Methods Mol Biol 1495:41-55
Lesniak, Wojciech G; Aboye, Teshome; Chatterjee, Samit et al. (2017) In vivo Evaluation of an Engineered Cyclotide as Specific CXCR4 Imaging Reagent. Chemistry 23:14469-14475
Gould, Andrew; Camarero, Julio A (2017) Cyclotides: Overview and Biotechnological Applications. Chembiochem 18:1350-1363
Camarero, Julio A (2017) Cyclotides, a versatile ultrastable micro-protein scaffold for biotechnological applications. Bioorg Med Chem Lett 27:5089-5099
Li, Yilong; Gould, Andrew; Aboye, Teshome et al. (2017) Full Sequence Amino Acid Scanning of ?-Defensin RTD-1 Yields a Potent Anthrax Lethal Factor Protease Inhibitor. J Med Chem 60:1916-1927
Borra, Radhika; Camarero, Julio A (2017) Protein Chemical Modification Inside Living Cells Using Split Inteins. Methods Mol Biol 1495:111-130
Li, Yilong; Aboye, Teshome; Breindel, Leonard et al. (2016) Efficient recombinant expression of SFTI-1 in bacterial cells using intein-mediated protein trans-splicing. Biopolymers 106:818-824
Aboye, Teshome; Meeks, Christopher J; Majumder, Subhabrata et al. (2016) Design of a MCoTI-Based Cyclotide with Angiotensin (1-7)-Like Activity. Molecules 21:152
Aboye, Teshome; Kuang, Yuting; Neamati, Nouri et al. (2015) Rapid parallel synthesis of bioactive folded cyclotides by using a tea-bag approach. Chembiochem 16:827-33

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