LAPSE:2023.6026
Published Article
LAPSE:2023.6026
High Throughput Expression Screening of Arabinofuranosyltransferases from Mycobacteria
February 23, 2023
Abstract
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. Arabinofuranosyltransferases 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 chromatography. 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.
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. Arabinofuranosyltransferases 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 chromatography. 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.
Record ID
Keywords
Arabinofuranosyltransferases, high-throughput protocol, membrane proteins, Mycobacteria, overexpression in E. coli, protein purification
Subject
Suggested Citation
Rodrigues J, Almeida VT, Rosário AL, Tan YZ, Kloss B, Mancia F, Archer M. High Throughput Expression Screening of Arabinofuranosyltransferases from Mycobacteria. (2023). LAPSE:2023.6026
Author Affiliations
Rodrigues J: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal [ORCID]
Almeida VT: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal [ORCID]
Rosário AL: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal
Tan YZ: Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, NY 10032, USA; National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY [ORCID]
Kloss B: Center on Membrane Protein Production and Analysis, New York Structural Biology Center, New York, NY 10027, USA [ORCID]
Mancia F: Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, NY 10032, USA [ORCID]
Archer M: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal [ORCID]
Almeida VT: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal [ORCID]
Rosário AL: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal
Tan YZ: Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, NY 10032, USA; National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY [ORCID]
Kloss B: Center on Membrane Protein Production and Analysis, New York Structural Biology Center, New York, NY 10027, USA [ORCID]
Mancia F: Department of Physiology and Cellular Biophysics, Columbia University Irving Medical Center, New York, NY 10032, USA [ORCID]
Archer M: Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa (ITQB NOVA), 2780-157 Oeiras, Portugal [ORCID]
Journal Name
Processes
Volume
9
Issue
4
First Page
629
Year
2021
Publication Date
2021-04-02
ISSN
2227-9717
Version Comments
Original Submission
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PII: pr9040629, Publication Type: Journal Article
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LAPSE:2023.6026
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External Link
https://doi.org/10.3390/pr9040629
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[v1] (Original Submission)
Feb 23, 2023
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Feb 23, 2023
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