# Over-expression, purification, and kinetic analysis of Mycobacterium tuberculosis WecA

## TL;DR

This study over-expressed and purified a tuberculosis-related enzyme, WecA, and analyzed its activity to support drug development.

## Contribution

A method for over-expressing and purifying a challenging membrane protein, WecA, from Mycobacterium tuberculosis is presented.

## Key findings

- WecA was successfully over-expressed using E. coli Lemo21(DE3) strain with T7 lysozyme regulation.
- Purified WecA was identified via mass spectrometry and its kinetic properties were analyzed.
- Tunicamycin was identified as a competitive inhibitor of WecA.

## Abstract

The N-acetylglucosamine-1-phosphate transferase (WecA)is a potential target for developing anti-tuberculosis drugs, due to its critical role in the synthesis of mycobacterial cell wall. The enzymatic study of WecA and the discovery of WecA inhibitors are therefore justified. However, WecA is a membrane protein with 11 transmembrane domains, making it difficult to be obtained, and even more difficult to perform activity studies. In order to gain sufficient WecA protein for activity investigation, the Escherichia coli (E. coli) Lemo21(DE3) strain was utilised in this study. The expression level of WecA was precisely regulated by T7 lysozyme. Purified WecA was obtained by affinity chromatography and identified by mass spectrometry. The kinetic properties of WecA were determined based on the detection of the product UMP. In addition, tunicamycin proved to be a competitive inhibitor. These results will lay theoretical foundations for the elucidation of WecA catalytic mechanism and the development of WecA inhibitors.