Abstract

The objective of this proposal is to demonstrate that naturally occurring photoproteins and their mutants can be used in the design and development of highly sensitive luminescent assays capable of small volume analysis. In particular, we propose to prepare and characterize new genetically engineered variants of aequorin, obelin, and other photoproteins, so that they are capable of emission of light at different wavelengths than those of the corresponding wild-type photoproteins. These photoproteins will be then employed as reagents in the development of assays for physiologically important molecules and for the high-throughput screening of biopharmaceuticals. By using the different variants of the photoproteins, array detection of biomolecules will be investigated. Research performed by our group during the current budget period demonstrated the usefulness of employing recombinant conjugates in the highly sensitive detection of biomolecules. We now propose to design and develop new methods based on the bioluminescence of photoproteins that are suitable for the microanalysis of important biomolecules, such as vitamins, drugs, peptides, etc., in nL-sized samples.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047915-08
Application #
6519504
Study Section
Special Emphasis Panel (ZRG1-BMT (01))
Program Officer
Edmonds, Charles G
Project Start
2000-03-01
Project End
2004-02-29
Budget Start
2002-03-01
Budget End
2003-02-28
Support Year
8
Fiscal Year
2002
Total Cost
$191,054
Indirect Cost

  Institution

Name
University of Kentucky
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
832127323
City
Lexington
State
KY
Country
United States
Zip Code
40506

  Publications

Zhang, Daohong; Broyles, David; Hunt, Eric A et al. (2017) A paper-based platform for detection of viral RNA. Analyst 142:815-823
Hunt, Eric A; Moutsiopoulou, Angeliki; Broyles, David et al. (2017) Expression of a soluble truncated Vargula luciferase in Escherichia coli. Protein Expr Purif 132:68-74
Yu, Xiaowen; Yang, Yu-Ping; Dikici, Emre et al. (2017) Beyond Antibodies as Binding Partners: The Role of Antibody Mimetics in Bioanalysis. Annu Rev Anal Chem (Palo Alto Calif) 10:293-320
Moutsiopoulou, Angeliki; Hunt, Eric; Broyles, David et al. (2017) Bioorthogonal Protein Conjugation: Application to the Development of a Highly Sensitive Bioluminescent Immunoassay for the Detection of Interferon-?. Bioconjug Chem 28:1749-1757
Head, Trajen; Dau, Peter; Duffort, Stephanie et al. (2017) An enhanced bioluminescence-based Annexin V probe for apoptosis detection in vitro and in vivo. Cell Death Dis 8:e2826
Mittal, Rahul; Debs, Luca H; Patel, Amit P et al. (2017) Neurotransmitters: The Critical Modulators Regulating Gut-Brain Axis. J Cell Physiol 232:2359-2372
Knecht, Leslie D; O'Connor, Gregory; Mittal, Rahul et al. (2016) Serotonin Activates Bacterial Quorum Sensing and Enhances the Virulence of Pseudomonas aeruginosa in the Host. EBioMedicine 9:161-169
Hunt, Eric A; Moutsiopoulou, Angeliki; Ioannou, Stephanie et al. (2016) Truncated Variants of Gaussia Luciferase with Tyrosine Linker for Site-Specific Bioconjugate Applications. Sci Rep 6:26814
Kumar, Manoj; Kovalski, Letícia; Broyles, David et al. (2016) Design and development of high bioluminescent resonance energy transfer efficiency hybrid-imaging constructs. Anal Biochem 498:1-7
Liu, Zhao-Jun; Daftarian, Pirouz; Kovalski, Letícia et al. (2016) Directing and Potentiating Stem Cell-Mediated Angiogenesis and Tissue Repair by Cell Surface E-Selectin Coating. PLoS One 11:e0154053

Showing the most recent 10 out of 20 publications