Title |
Enhancing the synthesizability of DP04 polymerase |
Publication Type |
thesis |
School or College |
School of Medicine |
Department |
Biochemistry |
Author |
Judd, David Paul |
Date |
2015-08 |
Description |
The total chemical synthesis of 361-residue Dpo4 DNA polymerase (including N-terminal His-tag) is an ambitious undertaking that pushes the limits of solid-phase peptide synthesis (SPPS). In chemical protein synthesis projects, smaller peptide segments are synthesized using SPPS and these segments are ligated together via native chemical ligation (NCL) to produce a full-length protein. NCL is a chemoselective reaction that requires a C-terminal thioester and an N-terminal Cys residue to ligate peptide segments. These requirements pose a challenge to synthetic protein chemists because Cys residues are rare in most proteins and C-terminal thioester selection is crucial to the success of the project because some thioesters react quickly and efficiently while others are slow and produce very low yields. A strategically placed mutation, usually to optimize ligation sites, can make an impossible chemical protein synthesis project possible. However, mutations must be chosen carefully to minimize their impact on the activity of the protein. In the case of Dpo4, we identified three positions where mutations would greatly simplify our chemical synthesis efforts. In order to safely make mutations, we used BLAST searches and crystal structure analysis to determine candidate residues. We recombinantly expressed mutated Dpo4 constructs and compared their activity against wild-type Dpo4. In this manuscript, we discuss the selection of the three mutated positions and the development of a PCR assay that allowed iv us to quantify differences in thermostability between mutant Dpo4 constructs and wild-type Dpo4. This research highlights the importance of strategically placed mutations in chemical protein synthesis projects and also has the potential to impact the biotechnology field with the introduction of mirror-image PCR using chemically synthesized D-Dpo4 as the DNA polymerase. |
Type |
Text |
Publisher |
University of Utah |
Subject |
Biochemistry |
Subject MESH |
Amino Acids; Amino Acid Sequence; Peptides; Ligands; DNA-Directed RNA Polymerases; DNA-Directed DNA Polymerase; Protein Engineering; Solid-Phase Synthesis Techniques; Molecular Chaperones; Protein Folding; Polymerase Chain Reaction; Mutation |
Dissertation Institution |
University of Utah |
Dissertation Name |
Master of Science |
Language |
eng |
Relation is Version of |
Digital version of Enhancing the Synthesizability of DP04 Polymerase |
Rights Management |
(c) David Paul Judd |
Format |
application/pdf |
Format Medium |
application/pdf |
Format Extent |
2,508,828 bytes |
Source |
Original in Marriott Library Special Collections |
ARK |
ark:/87278/s6sr3j6p |
Setname |
ir_etd |
ID |
1432973 |
Reference URL |
https://collections.lib.utah.edu/ark:/87278/s6sr3j6p |