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From 2014.igem.org

Overview

Stabilizing proteins is an incredibly important and time consuming task in the field of protein engineering. Current methods require using intimate knowledge of the protein to hypothesize point mutations that could possibly improve stability. Extensive in vitro testing follows involving cloning the new construct into your model organism, expressing the construct, purifying your protein of interest, and producing a melting curve to verify if indeed your mutation did improve stability. In addition to being time intensive, this method is unreliably successful.

Our team has developed a generalizable, high throughput method to select for the increased expression and stability of engineered proteins, making them more amenable to large-scale production in Escherichia coli and other downstream applications. Our method involves the insertion of proteins into a Gal4-VP16 transactivator that binds a promoter directly upstream of a GFP gene. The protein of interest is inserted into the middle of this complex using polypeptide linkers allowing for the subsequent selection of mutants associated with higher GFP output. We hypothesize that more stable proteins and their complexes will not be degraded by the cell’s natural machinery which will allow the Gal4-VP16 construct to produce higher levels of GFP. Less stable proteins will be degraded through natural mechanisms and will not produce GFP. The difference between these two populations can be evaluated using Flow Cytometry or Fluorescence Activated Cell Sorting.

^{Fig. 1. This novel method of protein stabilization is potentially generalizable, as it utilizes cells' natural pathways for maintaining protein folding, and high throughput when coupled with Fluorescence-Activated Cell Sorting (FACS).}

Our method utilizes degrons to produce the desired range of GFP based off of the proteins relative stability while applying a destabilizing influence to the protein complex. The degron illuminates the differences between proteins of varying stabilities. This allows the system to operate with a higher clarity between stable and unstable protein variants. While the degron exaggerates differences between stabilities, it also can be used as a variable tool that can be adjusted to fit your protein of interest. By placing the degron in different positions along the protein complex, you can impose different destabilizing effects on the construct.

The end goal of our project is to create a system that can be used in today’s protein engineering laboratories. By using Fluorescence Activated Cell Sorting of variants in a random mutagenesis library of our construct, we can sort out the highest GFP output variants which will correlate to the most stable variants. Through successive sorts the population will converge on a variant that improves stability of the protein complex. This revolutionary method is a generalizable alternative to current, labor intensive approaches for the selection of stable protein variants. Engineered proteins selected through this method could be produced in bacteria and aid in the development of thermostable, de novo protein therapeutics.