Abstract/Details

Mutagenesis studies and application of photoprotein aequorin


2010 2010

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Abstract (summary)

The photoprotein aequorin was originally isolated from jellyfish. Owing to its high sensitivity, low background and no toxicity, aequorin has recently widely used in biological and analytical applications, such as intracellular calcium detection, gene expression study, binding and immunoassay development, etc.

Recombinant aequorins can be easily obtained since the cDNA sequence of aequorin has been determined. Aequorin can be genetically conjugated to a target analyte to form a fusion protein which can be produced reproducibly and in a large amount. One immunoassay for angiotensin II detection using aequorin as a label has been developed. The proposed method, which utilized the aequorin-angiotensin II fusion protein, is simple and safe compared to conventional methods using radioisotopes, and is sensitive enough to estimate the angiotensin II levels in human plasma.

Aequorin and other calcium-binding photoproteins have similar bioluminescent properties such as similar maximum emission wavelengths and decay kinetics because all of them use coelenterazine as the substrate, which greatly limits its multiplexing capability (simultaneous detection of multiple analytes in a sample). To address this problem, two mutagenesis strategies, site-directed mutagenesis and random mutagenesis, coupled with different coelenterazine analogues, were used to get aequorin mutants that have improved bioluminescent properties.

First, several residues in the catalytic site of aequorin were selected for mutagenesis study by examining the X-ray crystal of the protein. The obtained mutants were characterized in terms of activity, half life, stability and spectral emission.

Next, error-prone PCR random mutagenesis was used to obtain several mutants with altered characteristics, especially two mutants with enhanced thermostability compared to wild type aequorin.

Finally, by combining a double mutation identified by our studies with another double mutation reported by another research group, a four-mutation mutant was constructed. The obtained mutant is much more thermostable than either of the two double mutants. Circular dichroism spectra of the four-mutation thermostable mutant were compared with those of wild type aequorin and the double mutant.

KEYWORDS: Photoprotein Aequorin, Bioluminescence Immunoassay Angiotensin II, Error-prone PCR, Mutagenesis

Indexing (details)


Subject
Molecular biology;
Analytical chemistry;
Biochemistry
Classification
0307: Molecular biology
0486: Analytical chemistry
0487: Biochemistry
Identifier / keyword
Pure sciences; Biological sciences; Aequorin; Angiotensin II; Immunoassay; Mutagenesis; Photoprotein
Title
Mutagenesis studies and application of photoprotein aequorin
Author
Qu, Xiaoge
Number of pages
139
Publication year
2010
Degree date
2010
School code
0102
Source
DAI-B 73/05, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
9781267184276
Advisor
Daunert, Sylvia
University/institution
University of Kentucky
University location
United States -- Kentucky
Degree
Ph.D.
Source type
Dissertations & Theses
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
3497379
ProQuest document ID
922660733
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
http://search.proquest.com/docview/922660733
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