High Throughput Expression Screening of Arabinofuranosyltransferases from Mycobacteria
Authors/Creators
- 1. processesArticle High Throughput Expression Screening of Arabinofuranosyltransferases from Mycobacteria José Rodrigues 1,† , Vanessa T. Almeida 1,† , Ana L. Rosário 1, Yong Zi Tan 2,3,‡ , Brian Kloss 4 , Filippo Mancia 2,* and Margarida Archer 1,* Citation: Rodrigues, J.; Almeida, V.T.; Rosário, A.L.; Tan, Y.Z.; Kloss, B.; Mancia, F.; Archer, M. High Throughput Expression Screening of Arabinofuranosyltransferases from Mycobacteria. Processes 2021, 9, 629. https://doi.org/10.3390/pr9040629 Academic Editor: Florian M. Wurm Received: 4 March 2021 Accepted: 26 March 2021 Published: 2 April 2021 Publisher's Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portuga
- 2. Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal
- 3. National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY 10027, USA
- 4. Center on Membrane Protein Production and Analysis, New York Structural Biology Center, New York, NY 10027, USA
- 5. Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, NY 10032, USA;
- 6. Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeira
Description
Studies on membrane proteins can help to develop new drug targets and treatments for
a variety of diseases. However, membrane proteins continue to be among the most challenging
targets in structural biology. This uphill endeavor can be even harder for membrane proteins from
Mycobacterium species, which are notoriously difficult to express in heterologous systems. Arabino-
furanosyltransferases are involved in mycobacterial cell wall synthesis and thus potential targets
for antituberculosis drugs. A set of 96 mycobacterial genes coding for Arabinofuranosyltransferases
was selected, of which 17 were successfully expressed in E. coli and purified by metal-affinity chro-
matography. We herein present an efficient high-throughput strategy to screen in microplates a large
number of targets from Mycobacteria and select the best conditions for large-scale protein production
to pursue functional and structural studies. This methodology can be applied to other targets, is cost
and time effective and can be implemented in common laboratories
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