title: Modular construction of multi-subunit protein complexes using engineered tags and microbial transglutaminase.
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 is 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.
authors.affiliation: Biological Sciences Graduate Program, - College of Computer, Mathematical and Natural Sciences, 4066 Campus Drive, University of Maryland, College Park, MD 20742, United States; Institute for Bioscience and Biotechnology Research, College Park, 5115 Plant Science and Landscape Architecture Building, University of Maryland, College Park, MD 20742, United States.
authors.forename: Narendranath
authors.initials: N
authors.lastname: Bhokisham
authors.affiliation: Fischell Department of Bioengineering, Room 3122, Jeong H. Kim Engineering Building (Bldg. #225), University of Maryland, College Park, MD 20742, United States.
authors.forename: Haig
authors.initials: H
authors.lastname: Pakhchanian
authors.affiliation: Institute for Bioscience and Biotechnology Research, College Park, 5115 Plant Science and Landscape Architecture Building, University of Maryland, College Park, MD 20742, United States; Fischell Department of Bioengineering, Room 3122, Jeong H. Kim Engineering Building (Bldg. #225), University of Maryland, College Park, MD 20742, United States.
authors.forename: David
authors.initials: D
authors.lastname: Quan
authors.affiliation: Institute for Bioscience and Biotechnology Research, College Park, 5115 Plant Science and Landscape Architecture Building, University of Maryland, College Park, MD 20742, United States; Fischell Department of Bioengineering, Room 3122, Jeong H. Kim Engineering Building (Bldg. #225), University of Maryland, College Park, MD 20742, United States.
authors.forename: Tanya
authors.initials: T
authors.lastname: Tschirhart
authors.affiliation: Institute for Bioscience and Biotechnology Research, College Park, 5115 Plant Science and Landscape Architecture Building, University of Maryland, College Park, MD 20742, United States; Fischell Department of Bioengineering, Room 3122, Jeong H. Kim Engineering Building (Bldg. #225), University of Maryland, College Park, MD 20742, United States.
authors.forename: Chen-Yu
authors.initials: CY
authors.lastname: Tsao
authors.affiliation: Institute for Bioscience and Biotechnology Research, College Park, 5115 Plant Science and Landscape Architecture Building, University of Maryland, College Park, MD 20742, United States; Fischell Department of Bioengineering, Room 3122, Jeong H. Kim Engineering Building (Bldg. #225), University of Maryland, College Park, MD 20742, United States.
authors.forename: Gregory F
authors.initials: GF
authors.lastname: Payne
authors.affiliation: Biological Sciences Graduate Program, - College of Computer, Mathematical and Natural Sciences, 4066 Campus Drive, University of Maryland, College Park, MD 20742, United States; Institute for Bioscience and Biotechnology Research, College Park, 5115 Plant Science and Landscape Architecture Building, University of Maryland, College Park, MD 20742, United States; Fischell Department of Bioengineering, Room 3122, Jeong H. Kim Engineering Building (Bldg. #225), University of Maryland, College Park, MD 20742, United States. Electronic address: bentley@umd.edu.
authors.forename: William E
authors.initials: WE
authors.lastname: Bentley
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000235:genetics; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics*; D011503:Transglutaminases / Q000737:chemistry / Q000235:genetics*
chemical_list: D000596:Amino Acids; D003432:Cross-Linking Reagents; D046912:Multiprotein Complexes; D021122:Protein Subunits; D011503:Transglutaminases
keywords: Biofabrication; Metabolon; Protein complex; Quorum sensing; Transglutaminase