Modular Construction of Multi-Subunit Protein Complexes using Engineered Tags and Microbial Transglutaminase.

Printer-friendly versionPrinter-friendly versionPDF versionPDF version
TitleModular Construction of Multi-Subunit Protein Complexes using Engineered Tags and Microbial Transglutaminase.
Publication TypeJournal Article
Year of Publication2016
AuthorsBhokisham, N, Pakhchanian, H, Quan, D, Tschirhart, T, Tsao, C-Y, Payne, GF, Bentley, WE
JournalMetab Eng
Date Published2016 May 26
ISSN1096-7184
Abstract

Motivations for the hierarchical assembly of protein complexes are diverse spanning biosensing, biomedical and bioreactor applications. The assembly processes should be simple, scalable, versatile, and biologically benign to minimize loss of component parts. A "plug and play" methodology comprising a generic linking apparatus may enable rapid design and optimization. One application that desires these qualities is metabolon construction wherein multiple enzymes are organized in defined pathways to mediate biochemical flux. Here, we propose a modular design by incorporation of crosslinking-compliant amino acid tags comprised of lysine or glutamine residues at the N- or C-termini of the to-be-assembled proteins. These amino acid tags enable covalent crosslinking using microbial transglutaminase (mTG). Modularity is demonstrated where stoichiometries and relative positions of enzymes and other functional proteins are altered. Construction of multifunctional complexes are demonstrated by crosslinking domains of different function and origin. Namely, we built a two-subunit quorum sensing (QS) biosynthetic metabolon on solid supports and altered stoichiometries of the limiting constituents to increase the overall rate of reaction. To display functionality beyond biosynthesis, we constructed a molecular communication 'device' (antibody binding Protein G-QS complex) to target bacterial cells and demonstrated tailored QS responses among targeted bacteria. We propose that this approach, solid phase mTG-mediated linkage of biological components, can be used for assembly within many environments including microreactors or lab-on-a-chip systems. Because the methodology is general, we envision construction of multi-functional protein complexes in a 'plug and play' fashion for a variety of biosensing and synthetic biology applications.

DOI10.1016/j.ymben.2016.05.004
Alternate JournalMetab. Eng.
PubMed ID27237361