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Updated: Aug 17, 2026
Production of Recombinant PRMT Proteins using the Baculovirus Expression Vector System
Published on: July 17, 2021
Expression and purification of a functionally active recombinant GDP-mannosyltransferase (PimA) from Mycobacterium
Xiaoling Gu1, Mao Chen, Qingzhong Wang
1State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, People's Republic of China.
Abstract:
Lipoarabinomannans (LAM), especially mannose-capped LAM, abundant in the cell wall of Mycobacterium tuberculosis (Mtb) exhibit a broad spectrum of immunomodulatory functions and emerge as key virulence factors that may be relevant drug targets. The pimA gene of mycobacteria encodes a alpha-mannosyltransferase involved in the transfer reaction of the very first mannose from GDP-mannose to the carrier lipid phosphatidyl-myo-inositol, a precursor in the synthesis of LAM. PimA has been proposed to play an essential role in the growth of mycobacteria. In this study, the pimA gene from M. tuberculosis H37Rv was cloned into the pET28a vector and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) strain, allowing the expression of the Mtb PimA in fusion with a histidine-rich peptide on the N-terminal. The Mtb PimA was purified from the supernatant of the lysed cells under native conditions by immobilized metal affinity chromatography. The purity and molecular weight of Mtb PimA were determined by high performance liquid chromatography and matrix-assisted laser desorption ionization time-of-flight. Circular dichroism spectroscopy study on Mtb PimA showed that the protein was folded. The enzyme assays revealed that Mtb PimA showed a requirement for Mg(2+) for the activity and the K(m) and V(max) values of Mtb PimA were estimated at 18 +/- 2 microM and 0.1 +/- 0.05 nmol/min/microg, respectively. This is the first report describing cloning and expression of GDP-mannosyltransferase gene of M. tuberculosis in E. coli.
Insights
Researchers cloned and expressed the Mycobacterium tuberculosis pimA gene in E. coli, yielding a functional alpha-mannosyltransferase essential for lipoarabinomannans (LAM) synthesis. This study provides a foundation for developing new tuberculosis drugs targeting LAM biosynthesis.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Lipoarabinomannans (LAM) are key virulence factors in Mycobacterium tuberculosis (Mtb), influencing host immune responses.
- The pimA gene encodes an alpha-mannosyltransferase crucial for the initial step in LAM synthesis.
Purpose of the Study:
- To clone and express the Mtb pimA gene in a heterologous system.
- To characterize the recombinant Mtb PimA protein and its enzymatic activity.
Main Methods:
- Cloning of Mtb pimA into the pET28a vector and expression in E. coli BL21 (DE3).
- Purification of Mtb PimA using immobilized metal affinity chromatography.
- Analysis of protein purity, molecular weight, folding, and enzymatic kinetics.
Main Results:
- Successful cloning, expression, and purification of recombinant Mtb PimA.
- Demonstration of Mtb PimA's folded structure via circular dichroism spectroscopy.
- Enzymatic assays revealed Mg(2+) dependency and kinetic parameters (Km = 18 ± 2 μM, Vmax = 0.1 ± 0.05 nmol/min/μg).
Conclusions:
- This study reports the first successful cloning and expression of the Mtb pimA gene in E. coli.
- The characterized Mtb PimA enzyme is active and requires Mg(2+), providing insights into LAM biosynthesis.
- This work lays the groundwork for further investigation of Mtb PimA as a potential drug target.
