Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


20140826 Thesis by JoongseokOh.pdf (1.23 MB)

ETD Abstract Container

Abstract Header

DESIGN OF RECOMBINANT TENEBRIO MOLITOR ANTIFREEZE PROTEIN FOR PURIFICATION USING ELASTIN-LIKE POLYPEPTIDE TAG

OH, JOONGSEOK
http://rave.ohiolink.edu/etdc/view?acc_num=csu1409230499

Abstract Details

2014, Master of Science in Biomedical Engineering, Cleveland State University, Washkewicz College of Engineering.
Fusion protein technologies can aid to improve solubility of recombinant protein from microorganisms and help recombinant protein purification. Elastin-like polypeptides (ELP) as a fusion tag can be utilized to facilitate the purification of recombinant proteins because ELP can provide its thermally responsive behavior to ELP tagged proteins. An ELP tag can be used as the purification carrier through inverse transition cycling (ITC), which is a simple and non chromatographic separation process. The purification through ITC can reduce cost and can be quickly performed compared to other purification methods. However, we further considered ELP tag removal because it may possibly hinder the structure and function of fusion partners (or target proteins). Tenebrio molitor antifreeze protein (TmAFP) is an insect AFP and it is classified as a hyperactive antifreeze protein because its activity is 10 to 100 times greater than AFPs from other sources. TmAFP as a target protein has been purified by cold finger purification or chromatographic separation but there are drawbacks. The overall goal of this work is to design and implement a strategy for high yield of recombinant TmAFP in E. coli through purification using ELP tags. This includes ELP tagged TmAFP with a TEV cleavage site. DNA encoding this tag was designed and successfully added to the N-terminus of the TmAFP gene. Then the ELP tags were added to the N-terminus of TmAFP and ELP tag DNA was successfully added to N-terminus of TEV protease. Of note, ELP[(GVGVP)40] tagged DNA was successfully added to the N-terminus TEV protease but ELP[(GVGVP)20] tagged DNA was not successfully added. Then the ELP tagged fusion proteins were successfully expressed. ELP tagged TmAFPs were successfully purified by using ITC while ELP tagged TEV protease was not successfully purified by ITC.
Nolan Holland, PhD (Advisor)
Joanne Belovich, PhD (Committee Member)
Xue-Long Sun, PhD (Committee Member)
90 p.
Biomedical Engineering

Recommended Citations

Citations

  • OH, J. (2014). DESIGN OF RECOMBINANT TENEBRIO MOLITOR ANTIFREEZE PROTEIN FOR PURIFICATION USING ELASTIN-LIKE POLYPEPTIDE TAG [Master's thesis, Cleveland State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=csu1409230499

    APA Style (7th edition)

  • OH, JOONGSEOK. DESIGN OF RECOMBINANT TENEBRIO MOLITOR ANTIFREEZE PROTEIN FOR PURIFICATION USING ELASTIN-LIKE POLYPEPTIDE TAG. 2014. Cleveland State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=csu1409230499.

    MLA Style (8th edition)

  • OH, JOONGSEOK. "DESIGN OF RECOMBINANT TENEBRIO MOLITOR ANTIFREEZE PROTEIN FOR PURIFICATION USING ELASTIN-LIKE POLYPEPTIDE TAG." Master's thesis, Cleveland State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=csu1409230499

    Chicago Manual of Style (17th edition)

csu1409230499
786
© 2014, all rights reserved.
This open access ETD is published by Cleveland State University and OhioLINK.