Directed evolution improves the catalytic efficiency of TEV protease

There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

Abstract

Tobacco etch virus protease (TEV) is one of the most widely-used proteases in biotechnology because of its exquisite sequence-specificity. A limitation, however, is its slow catalytic rate. We developed a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is read out via proteolytic release of a membrane-anchored transcription factor, and we temporally regulate access to TEV’s cleavage substrate using a photosensory LOV domain. By gradually decreasing light exposure time, we enriched faster variants of TEV over multiple rounds of selection. Our S153N mutant (uTEV1Δ), when incorporated into the calcium integrator FLARE, improved the signal/background ratio by 27-fold, and enabled recording of neuronal activity in culture with 60-second temporal resolution. Given the widespread use of TEV in biotechnology, both our evolved TEV mutants and the directed evolution platform used to generate them, could be beneficial across a wide range of applications.

Related collections

Most cited references 33

Record: found
Abstract: found
Article: not found

Directed evolution of APEX2 for electron microscopy and proteomics

Stephanie Lam, Jeffrey D. Martell, Kimberli J. Kamer … (2014)

APEX is an engineered peroxidase that functions both as an electron microscopy tag, and as a promiscuous labeling enzyme for live-cell proteomics. Because the limited sensitivity of APEX precludes applications requiring low APEX expression, we used yeast display evolution to improve its catalytic efficiency. Our evolved APEX2 is far more active in cells, enabling the superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins and the use of electron microscopy to resolve the sub-mitochondrial localization of calcium uptake regulatory protein MICU1.

0 comments Cited 488 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9.

Lukasz J. Swiech, Matthias Heidenreich, Abhishek Banerjee … (2015)

Probing gene function in the mammalian brain can be greatly assisted with methods to manipulate the genome of neurons in vivo. The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) can be used to edit single or multiple genes in replicating eukaryotic cells, resulting in frame-shifting insertion/deletion (indel) mutations and subsequent protein depletion. Here, we delivered SpCas9 and guide RNAs using adeno-associated viral (AAV) vectors to target single (Mecp2) as well as multiple genes (Dnmt1, Dnmt3a and Dnmt3b) in the adult mouse brain in vivo. We characterized the effects of genome modifications in postmitotic neurons using biochemical, genetic, electrophysiological and behavioral readouts. Our results demonstrate that AAV-mediated SpCas9 genome editing can enable reverse genetic studies of gene function in the brain.

0 comments Cited 325 times – based on 0 reviews      Review now

Bookmark

Record: found
Abstract: found
Article: not found

Engineered ascorbate peroxidase as a genetically-encoded reporter for electron microscopy

Jeffrey D. Martell, Thomas J. Deerinck, Yasemin Sancak … (2012)

Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments 1 or require light and are difficult to use 2 . Here we report the development of a simple and robust EM genetic tag, called “APEX,” that is active in all cellular compartments and does not require light. APEX is a monomeric 28 kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins. We also fused APEX to the N- or C-terminus of the mitochondrial calcium uniporter (MCU), a newly identified channel whose topology is disputed 3,4 . MCU-APEX and APEX-MCU give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N-and C-termini of MCU face the matrix.