Publication

Transforming a (beta/alpha)8-Barrel Enzyme into a Split-Protein Sensor through Directed Evolution

Kai Johnsson
2004
Journal Articles

Abstract

Split-protein sensors have become an important tool for the anal. of protein-protein interactions in living cells. We present here a combinatorial method for the generation of new split-protein sensors and demonstrate its application toward the (b/a)8-barrel enzyme N-(5'-phosphoribosyl)-anthranilate isomerase Trp1p from Saccharomyces cerevisiae. The generated split-Trp protein sensors allow for the detection of protein-protein interactions in the cytosol as well as the membrane by enabling trp1 cells to grow on medium lacking tryptophan. This powerful selection complements the repertoire of the currently used split-protein sensors and provides a new tool for high-throughput interaction screening. [on SciFinder (R)]

Official source

https://infoscience.epfl.ch/entities/publication/e2f7dbbd-6c2b-49ba-833b-4cf983fec2cf

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Kai Johnsson
2004
Journal Articles

Abstract

Official source

https://infoscience.epfl.ch/entities/publication/e2f7dbbd-6c2b-49ba-833b-4cf983fec2cf

About this result

Related concepts (32)

Two-hybrid screening

Two-hybrid screening (originally known as yeast two-hybrid system or Y2H) is a molecular biology technique used to discover protein–protein interactions (PPIs) and protein–DNA interactions by testing for physical interactions (such as binding) between two proteins or a single protein and a DNA molecule, respectively. The premise behind the test is the activation of downstream reporter gene(s) by the binding of a transcription factor onto an upstream activating sequence (UAS).

Protein–protein interaction

Protein–protein interactions (PPIs) are physical contacts of high specificity established between two or more protein molecules as a result of biochemical events steered by interactions that include electrostatic forces, hydrogen bonding and the hydrophobic effect. Many are physical contacts with molecular associations between chains that occur in a cell or in a living organism in a specific biomolecular context. Proteins rarely act alone as their functions tend to be regulated.

Directed evolution

Directed evolution (DE) is a method used in protein engineering that mimics the process of natural selection to steer proteins or nucleic acids toward a user-defined goal. It consists of subjecting a gene to iterative rounds of mutagenesis (creating a library of variants), selection (expressing those variants and isolating members with the desired function) and amplification (generating a template for the next round). It can be performed in vivo (in living organisms), or in vitro (in cells or free in solution).

Related publications (2)

Transforming a (beta/alpha)8-Barrel Enzyme into a Split-Protein Sensor through Directed Evolution

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