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Selective Fusion-Tag-Catalyzed Protein Immobilizations for Microarray and Biosensor Applications

Voelker, Alden Earl
http://rave.ohiolink.edu/etdc/view?acc_num=case1370448393

Abstract Details

2013, Doctor of Philosophy, Case Western Reserve University, Chemistry.
Protein microarrays are becoming increasingly popular as platforms for applications ranging from bioanalyte detection (biosensors and diagnostics) to enzyme activity profiling and even the biosynthesis of natural products and their analogs in vitro. Selective and efficient protein immobilization methods are essential for such applications, and herein we describe the first example of such an immobilization that exploits the self-catalysis and binding properties of the commonly-used glutathione transferase (GST) purification tag. A library of 25 analogs of the known GST substrate 1-chloro-2,4-dinitrobenzene (CDNB) was synthesized, and 20 of these compounds bearing bioorthogonal linker substituents were screened for glutathione (GSH) conjugation activity with the GST from the helminth worm Schistosoma japonicum. The Michaelis-Menten kinetics for several enzymatic reactions were studied, and it was observed that improved aqueous solubility and strongly electron-withdrawing ring substituents accelerate the conjugation reaction, providing hints for the design of a second generation of GST substrates. The regiochemistry of the SNAr reaction was also studied; it was found that conjugation of GSH is favored at the 4-position of the aryl ring, a result that was rationalized by noting the increased electrophilicity of that position due to resonance effects. An alkyne-bearing CDNB analog was chosen to serve as a linker for the immobilization of SjGST on an azide-functionalized glass microscope slide. A “click” reaction was performed to modify the surface with the CDNB analog, and subsequent treatment with SjGST and GSH led to an array of immobilized protein that was characterized via contact-angle microscopy, fluorescence immunolabeling, and atomic force microscopy. Arrays of SjGST fabricated in this way showed an 18-fold higher fluorescence detection signal compared to non-oriented proteins, and a 6-fold increase in signal versus conventionally-immobilized GSH. As a first step toward the creation of a biosynthetic microarray, a GST-tagged isonitrile synthase, IsnA, was cloned, expressed, and immobilized using this technique. Future experiments will be directed towards the creation of a functional biosynthetic chip that can produce several cyanobacterial natural products of interest to our lab.
Rajesh Viswanathan (Advisor)
Michael Zagorski (Committee Chair)
Mary Barkley (Committee Member)
Gregory Tochtrop (Committee Member)
John Mieyal (Committee Member)
305 p.
Biochemistry; Chemistry; Organic Chemistry
microarrays; GST-tag; fusion proteins; SjGST; CDNB; protein modification; protein immobilization; protein biochip

Recommended Citations

Citations

  • Voelker, A. E. (2013). Selective Fusion-Tag-Catalyzed Protein Immobilizations for Microarray and Biosensor Applications [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1370448393

    APA Style (7th edition)

  • Voelker, Alden. Selective Fusion-Tag-Catalyzed Protein Immobilizations for Microarray and Biosensor Applications. 2013. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1370448393.

    MLA Style (8th edition)

  • Voelker, Alden. "Selective Fusion-Tag-Catalyzed Protein Immobilizations for Microarray and Biosensor Applications." Doctoral dissertation, Case Western Reserve University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=case1370448393

    Chicago Manual of Style (17th edition)

case1370448393
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© 2013, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.