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Selecting for Fast Protein-Protein Association As Demonstrated on a Random TEM1 Yeast Library Binding BLIP.

Abstract：

:Protein-protein interactions mediate the vast majority of cellular processes. Though protein interactions obey basic chemical principles also within the cell, the in vivo physiological environment may not allow for equilibrium to be reached. Thus, in vitro measured thermodynamic affinity may not provide a complete picture of protein interactions in the biological context. Binding kinetics composed of the association and dissociation rate constants are relevant and important in the cell. Therefore, changes in protein-protein interaction kinetics have a significant impact on the in vivo activity of the proteins. The common protocol for the selection of tighter binders from a mutant library selects for protein complexes with slower dissociation rate constants. Here we describe a method to specifically select for variants with faster association rate constants by using pre-equilibrium selection, starting from a large random library. Toward this end, we refine the selection conditions of a TEM1-β-lactamase library against its natural nanomolar affinity binder β-lactamase inhibitor protein (BLIP). The optimal selection conditions depend on the ligand concentration and on the incubation time. In addition, we show that a second sort of the library helps to separate signal from noise, resulting in a higher percent of faster binders in the selected library. Fast associating protein variants are of particular interest for drug development and other biotechnological applications.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Cohen-Khait R,Schreiber G

doi

10.1021/acs.biochem.8b00172

subject

Has Abstract

pub_date

2018-08-07 00:00:00

pages

4644-4650

issue

31

eissn

0006-2960

issn

1520-4995

journal_volume

57

pub_type

杂志文章

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