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Modification, Expression, and Purification of Hyperactive Antifreeze Proteins from Insect Tenebrio Molitor

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2011, Master of Science in Biomedical Engineering, Cleveland State University, Fenn College of Engineering.

Antifreeze proteins (AFPs), also known as thermal hysteresis proteins, can protect organisms from damage caused by freezing by lowering the freezing point of fluids. AFPs can bind to small ice crystals, inhibit ice growth, and change the morphology of ice crystals. The difference between the freezing temperature and the melting temperature is termed thermal hysteresis (TH). There are different types of AFPs, such as fish AFPs (type I, II, III, and IV) and insect AFPs. These AFPs can also be classified by their thermal hysteresis activity: moderately active AFPs and hyperactive AFPs. Previous research groups successfully constructed AFP (type I, and type III) multimers, and the thermal hysteresis measurement showed that the size of multimers can affect TH activities. However, how the TH of insect AFPs will be affected by multimerization is still unknown, since they already have much higher thermal hysteresis activities than fish AFPs in nature. Making active multimers of insect AFPs can be harder than type I and type III fish AFPs because of their highly disulfide-bonded structure.

The aim of this thesis is to construct trimers of Tenebio molitor (TmAFP) by adding a foldon domain. The foldon domain has been added separately to N-teminus and C-terminus of TmAFP. Cold finger purification was utilized for the protein purification process. The purified proteins were then run on protein gels, demonstrating that trimers of TmAFP were successfully made, for both the N-terminus and the C-terminus foldon domains; the TmAFPs were active, indicating proper folding.

Nolan Holland, PhD (Advisor)
Dhananjai Shah, PhD (Committee Member)
Rolf Lustig, PhD (Committee Member)
67 p.

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Citations

  • Bu, S. (2011). Modification, Expression, and Purification of Hyperactive Antifreeze Proteins from Insect Tenebrio Molitor [Master's thesis, Cleveland State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=csu1328667906

    APA Style (7th edition)

  • Bu, Sen. Modification, Expression, and Purification of Hyperactive Antifreeze Proteins from Insect Tenebrio Molitor. 2011. Cleveland State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=csu1328667906.

    MLA Style (8th edition)

  • Bu, Sen. "Modification, Expression, and Purification of Hyperactive Antifreeze Proteins from Insect Tenebrio Molitor." Master's thesis, Cleveland State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=csu1328667906

    Chicago Manual of Style (17th edition)