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Protein engineering for novel protein-protein interactions and protein labeling
Protein-protein interactions (PPIs) are essential in many biological processes, such as cell cycle, metabolic engineering, and environment sensing. The function of a protein can be controlled or mediated by the association of another protein. In Chapter 1, a short review on PPIs was given, focusing on the biological importance of PPIs and tools that are commonly used to manipulate PPIs. In the subsequent chapter, cellulosome, a naturally occurring multi-enzyme complex for efficient degradation of cellulose, was studied. The chimeric structure of cellulosome is mediated by the cohesin and dockerin interaction. In our study, we sought to achieve the regio- and stoichiometric controlled assembly of designer cellulosomes through engineering orthogonal cohesin-dockerin pairs. A URA3/5-FOA counter selection system was modified in E. coli, presenting the first example of this negative selection method in bacterial two-hybrid system. It allows the selection of orthogonal protein interaction pairs. The limited number of naturally available cohesin-dockerin pairs poses a major obstacle for the controlled assembly of designer cellulosome containing larger number of different catalytic subunits. Successful expansion of the repertoire of orthogonal cohesin-dockerin pairs in the present study will facilitate the generation of synthetic cellulosome(s) that can catalyze the degradation of plant cellulosic materials more efficiently, thus, have attractive promise to be applied as valuable tools to generate biomass-derived feedstocks for biofuel and bioenergy production. In Chapter 3, the barnase-bastar pair was used as a second model for evolving orthogonal protein pairs with our two-hybrid selection system. The interacting interface of the protein pair was randomized by site-saturation mutagenesis. An orthogonal barstar mutant was identified. The barnase-barstar pair has great potential in the nanomaterial field for drug delivery and therapeutics. The orthogonal barnase-barstar protein pair will provide more diversity for nanomaterial research. Genetic code expansion allows us to site-specifically introduce the unnatural amino acids with diverse chemical functional groups into the protein. It could be applied in many areas, such as trapping protein-protein interactions, conducting protein labeling, and controlling enzyme activities. In Chapter 4, a photoactivatable SH2 domain was constructed. A photo-caged amino acid was incorporated into the pTyr-binding site of an SH2 domain. The interaction between this SH2 domain mutant and its peptide substrate could be turned on upon UV irradiation. In Chapter 5, a styrene-derived lysine amino acid (KStyr) was site-specifically and efficiently incorporated into proteins in both E. coli and mammalian cells. The fluorogenic reaction between KStyr and tetrazine was used to label proteins both in vitro and in vivo.
Song, Xi, "Protein engineering for novel protein-protein interactions and protein labeling" (2016). ETD collection for University of Nebraska - Lincoln. AAI10102321.