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Shakalli_Dissertation.pdf (2.23 MB)
ETD Abstract Container
Abstract Header
Applied Protein Engineering for Bacterial Biosensor and Protein Purification
Author Info
Shakalli, Miriam Joan
Permalink:
http://rave.ohiolink.edu/etdc/view?acc_num=osu1450805485
Abstract Details
Year and Degree
2016, Doctor of Philosophy, Ohio State University, Chemical Engineering.
Abstract
Since the 1980s, better understanding of protein structure and function has allowed for the design of useful proteins and their subsequent modification. In our laboratory, advances in protein engineering have been harnessed for the development of biosensors and the optimization of bioseparations. This dissertation encompasses work on both of these applications. First, previous work in our laboratory has developed a simple reporter protein in
E. coli
that can sense hormone-mimicking compounds of human nuclear hormone receptors (NHRs) and report their presence and activity through changes in growth phenotype. This system was extended from our current human targets to incorporate an insect NHR. The nuclear hormone receptor HR96 of both
Aedes aegypti
and
Tribolium castaneum
were designed, constructed, and optimized to validate novel bacterial biosensors for the detection of potential insecticides. This cell-based system was then incorporated into a cell-free expression environment to establish a faster colorimetric biosensing assay. Preliminary results show that our bacterial biosensor can be expressed in a cell-free protein synthesis reaction without compromising the sensitivity and selectivity of its response to known ligands. Second, our laboratory seeks to optimize the self-cleaving intein tag as a potential purification platform technology. We have previously shown that the combination of the elastin-like polypeptide (ELP) as a self-aggregating purification tag and the ΔI-CM intein can facilitate the purification of a target protein. This methodology not only eliminates the need of expensive chromatography resins but also eliminates the expenses associated with the use of proteases to recover the native protein after its purification. This promising tool was studied further to determine the optimal ELP tag length required to maximize both expression and purification yields. The shorter ELP tag lengths improved protein expression levels of three model target proteins. However, the purification yield was not favorable. Longer ELP tag lengths resulted in higher purification recoveries relative to the amount of protein expressed.
Committee
David Wood (Advisor)
James Rathman (Committee Member)
Jeffrey Chalmers (Committee Member)
Pages
142 p.
Subject Headings
Chemical Engineering
Keywords
intein
;
biosensors
;
protein purification
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Citations
Shakalli, M. J. (2016).
Applied Protein Engineering for Bacterial Biosensor and Protein Purification
[Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1450805485
APA Style (7th edition)
Shakalli, Miriam.
Applied Protein Engineering for Bacterial Biosensor and Protein Purification.
2016. Ohio State University, Doctoral dissertation.
OhioLINK Electronic Theses and Dissertations Center
, http://rave.ohiolink.edu/etdc/view?acc_num=osu1450805485.
MLA Style (8th edition)
Shakalli, Miriam. "Applied Protein Engineering for Bacterial Biosensor and Protein Purification." Doctoral dissertation, Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1450805485
Chicago Manual of Style (17th edition)
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Document number:
osu1450805485
Download Count:
439
Copyright Info
© 2016, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.