Date of Award
Doctor of Philosophy
Eric T. Boder
Bamin Khomami, Paul D. Frymier, Christopher Stephens
There is a great interest for protein immobilization and generating protein three-dimensional assemblies in nano-biotechnology. The main challenge in such protein immobilization and oligomerization is stability and lack of control over the protein ligation site. An enzymatic method overcomes these issues by site-specific protein ligation using conditions compatible with protein structure and stability. Sortase A, a transpeptidase that naturally binds threonine in a LPXTG sequence and glycine in a GGG sequence, provides a covalent site-specific protein immobilization and ligation tool that can be used in generating site-specific protein 3D assemblies. Sortase A accepts various nucleophile substrates. Therefore, engineering Sortase A substrate specificity to create a more powerful tool to site-specifically ligate proteins together and to the surface is advantageous. Toward the goal of generating protein 3D assemblies and engineering Sortase A, we accomplished the following:
First, we site-specifically immobilized recombinant fluorescent proteins on the surface using sortase reaction. A self-assembled layer of GGGC or AAAC peptides, S. aureus or S. pyogenes Sortase nucleophile substrates, were made on the gold surface. Recombinant fluorescent proteins with LPETG/A tag at the C-terminus were then immobilized on this layer using corresponding sortase A reactions.
We then generated protein wires and assemblies in solution and on the surface in a controlled manner using two strategies. In the first strategy, we used two orthogonal sortases with a slightly different substrate specificity, S. pyogenes and S. aureus sortases, to control the protein assembly. In the second strategy, we used an enterokinase-cleavable protecting sequence at the N-terminus of a bifunctional protein, such that enterokinase-mediated removal will activate the N-terminus as a nucleophile substrate after each round of sortase-mediated immobilization and enable sequential deposition of single layers.
As the last goal of this study, we used yeast surface display and directed evolution to engineer Sortase A substrate specificity toward primary amine in the side chain of lysine in a pilin box sequence. A library of sortase mutants and LPETG substrate were displayed on the yeast surface. Sortase library was screened using FACS and mutants with the most activity toward the pilin box sequence were selected and analyzed.
Raeeszadeh-Sarmazdeh, Maryam, "Engineering Sortase A for generating site-specific protein 3D assemblies. " PhD diss., University of Tennessee, 2014.