Difference between revisions of "Team:Heidelberg/Protein Interaction"
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For many enzymes auto-reassembly split sites are known, however the efficiency of the joined fragments usually does not reach wildtype efficiency, as it was shown for Cas9 enzymes (Zetsche et al, Nature Biotechnology, 2015; Kaya et al, Plant Cell Physiol, 2017) Consequently, a directed evolution of split enzymes that enhance auto-reassembly is of great interest for the synthetic biology community. To demonstrate that an improved protein-protein interaction is not only possible for the Bt toxin, we used a split T7-polymerase as another example (Tiun Han et al, ACS Synthetic Biolog, 2017). We aimed at enhancing the auto-reassembly efficiency of different split sites using two in our lab established methods, PACE and PREDCEL (phage-related discontinuous evolution), which allow a fast and relatively easy directed evolution. | For many enzymes auto-reassembly split sites are known, however the efficiency of the joined fragments usually does not reach wildtype efficiency, as it was shown for Cas9 enzymes (Zetsche et al, Nature Biotechnology, 2015; Kaya et al, Plant Cell Physiol, 2017) Consequently, a directed evolution of split enzymes that enhance auto-reassembly is of great interest for the synthetic biology community. To demonstrate that an improved protein-protein interaction is not only possible for the Bt toxin, we used a split T7-polymerase as another example (Tiun Han et al, ACS Synthetic Biolog, 2017). We aimed at enhancing the auto-reassembly efficiency of different split sites using two in our lab established methods, PACE and PREDCEL (phage-related discontinuous evolution), which allow a fast and relatively easy directed evolution. | ||
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Figure One: Selecting for efficient T7-RNAP auto-reassembly in directed evolution experiment| | Figure One: Selecting for efficient T7-RNAP auto-reassembly in directed evolution experiment| | ||
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Revision as of 07:12, 1 November 2017
Protein Interaction
Improve Protein Reassembly
Introduction
Protein Interactions in Directed Evolution Context
The proteome reflects the entirety of proteins that is expressed by a cell or an organism. Most of these proteins do not function alone. Instead, they are dependent on the interactions with a multitude of other proteins to fulfill their functions within the cell. Protein-protein interactions are responsible for almost any kind of cellular processes, such as signal transduction, cell-cell contact, transport, metabolism, cell motion or even antigen-antibody recognition (Larance, Nature Reviews Molecular Cell Biology, 2015). These interactions are enabled by electrostatic forces due to the different chemical characteristics of the amino acids. Nowadays, many biochemical or microscopy-based methods are available to investigate protein-protein interactions, for instance protein complex immunoprecipitation (Co-IP), Förster resonance energy transfer (FRET) or yeast two-hybrid screenings. As easy it is to detect a protein-protein interaction, as difficult it is to alter the strength of these interactions, although the magnitude of interaction can influence the cellular outcome, for example a stronger signal transduction or a higher expression rate. Phage-assisted continuous evolution (PACE) allows a directed evolution of different kinds of proteins within a few days as it was for example shown for RNA polymerases, proteases or aminoacyl-tRNA synthetases. (Esvelt et al, Nature, 2011; Dickinson et al, Nat Commui., 2014; Bryson et al, Nat Chem Biol. 2017). Technically, a directed evolution towards a tighter protein-protein interaction is possible as well, as it was shown for the toxin Cry1Ac from Bacillus thuringiensis (Bt toxin) that binds a cadherin-like receptor (Badran et al, Nature, 2016). A special type of a protein-protein interaction are split enzymes that allow an auto-reassembly. They usually consist of two enzyme fragments (an N‑terminal and C‑terminal fragment) that are expressed separately, and fuse post translation. This protein fragment complementation is advantageous for any kind of application where the size of an expression cassette is a limiting factor, for instance for the packaging of DNA into virus capsids.Motivation
For many enzymes auto-reassembly split sites are known, however the efficiency of the joined fragments usually does not reach wildtype efficiency, as it was shown for Cas9 enzymes (Zetsche et al, Nature Biotechnology, 2015; Kaya et al, Plant Cell Physiol, 2017) Consequently, a directed evolution of split enzymes that enhance auto-reassembly is of great interest for the synthetic biology community. To demonstrate that an improved protein-protein interaction is not only possible for the Bt toxin, we used a split T7-polymerase as another example (Tiun Han et al, ACS Synthetic Biolog, 2017). We aimed at enhancing the auto-reassembly efficiency of different split sites using two in our lab established methods, PACE and PREDCEL (phage-related discontinuous evolution), which allow a fast and relatively easy directed evolution.
Figure One: Selecting for efficient T7-RNAP auto-reassembly in directed evolution experiment
This is how Selection works...
