Research Article: ESCORTing proteins directly from whole cell-lysate for single-molecule studies

Date Published: October 15, 2017

Publisher: Elsevier

Author(s): Shwetha Srinivasan, Jagadish P. Hazra, Gayathri S. Singaraju, Debadutta Deb, Sabyasachi Rakshit.

http://doi.org/10.1016/j.ab.2017.07.022

Abstract

We have developed a method for Enzymatic Sortase-assisted Covalent Orientation-specific Restraint Tethering (ESCORT) recombinant proteins onto surfaces directly from cell-lysate. With an improved surface passivation method, we obviate the cumbersome purification steps even for single molecule studies that demand high purity in the sample. We demonstrated high-specificity of the method, high-passivity of the surface and uncompromised functional integrity of anchored proteins using single molecule fluorescence and force-mapping. We anticipate that this method will substantially reduce the investment by way of time, money and energy in the area of single molecule studies.

Partial Text

Interaction of proteins with biological and chemical molecules plays an important role in various cellular biosynthetic and degradation pathways controlling major processes in living organisms [1]. Quantitative understanding of these interactions is of primary importance to gain insight into different basic biochemical processes happening inside a living organism.

G.S.S. and S.R. conceived the idea for the project. S.S. and J.P.H. performed the experiments. G.S.S. has contributed in the force-mapping using AFM. S.S. and S.R. prepared the manuscript. G.S.S. and D.D. expressed and purified Sortase A, Cdh23 and Pcdh15.

Authors declare no competing financial interests.

 

Source:

http://doi.org/10.1016/j.ab.2017.07.022

 

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