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Protein Engineering Hydrophobic Core Residues of Computationally Designed Protein G and Single-Chain Rop: Investigating the Relationship between Protein Primary structure and Protein Stability through High-Throughput Approaches

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2014, Master of Science, Ohio State University, Chemistry.
The sequence-structure-stability relationship is a key problem in the field of protein science. Although a large amount of research has been working on it in various methods and aspects, it is still not completely understood. Recently, the cooperation between rational design and combinatorial library methods bring new insight into the protein hydrophobic core. In this study, we investigated the influence of hydrophobic core residue packing to protein stability according to a computationally designed Protein G homolog and a single-chain four helix-bundle protein Rop. Based on a previously computationally designed protein G, we established two parallel hydrophobic core libraries -muti-site and single-site-with 6 residues in the hydrophobic core randomized simultaneously or individually. High Throughput Thermal Scanning (HTTS) and colony-based DNA sequencing were utilized to investigate the protein thermal stability. The comparison of the HTTS from the two libraries indicated that none of the expected mutations results in a thermally more stable protein than the computationally designed protein G, and the single-site mutation showed a similar effect than multi-site mutation on the computationally designed protein G. The original computational design was more stable than both of the two library designs. The core library of single-chain Rop was constructed as the eight residues in the central two layers randomized into all 20 amino acids. The large library was screened for Rop function, based on a cell-based screen with GFP reporter plasmid. The library was selected to eliminate the background inactive Rop protein and enrich the active Rop, and maximally cover the library size. Unfortunately, a significant amount of the colony-based DNA sequencing results showed the presence of non-authentic Rop sequence. Cloning contamination and arabinose concentration used in screening could be two potential factors that cause the failure of screening and selection. The library construction and cloning procedure also need to be revisited.
Thomas Magliery (Advisor)
Karin Musier-Forsyth (Committee Member)
62 p.

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Citations

  • Li, W. (2014). Protein Engineering Hydrophobic Core Residues of Computationally Designed Protein G and Single-Chain Rop: Investigating the Relationship between Protein Primary structure and Protein Stability through High-Throughput Approaches [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1398956266

    APA Style (7th edition)

  • Li, Weiyi. Protein Engineering Hydrophobic Core Residues of Computationally Designed Protein G and Single-Chain Rop: Investigating the Relationship between Protein Primary structure and Protein Stability through High-Throughput Approaches. 2014. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1398956266.

    MLA Style (8th edition)

  • Li, Weiyi. "Protein Engineering Hydrophobic Core Residues of Computationally Designed Protein G and Single-Chain Rop: Investigating the Relationship between Protein Primary structure and Protein Stability through High-Throughput Approaches." Master's thesis, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1398956266

    Chicago Manual of Style (17th edition)