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Updated: Jul 18, 2026
Mycobacterium tuberculosis Extracellular Vesicle Enrichment through Size Exclusion Chromatography
Published on: May 19, 2022
[Expression and purification of Rv3369 of Mycobacterium tuberculosis]
Chong Lv1, Xin Jiang, Xiao-Ling Gu
1State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China. lvchong0426@sohu.com
Abstract:
To obtain purified recombinant Rv3369 protein by means of expressing the Rv3369 protein of Mycobacterium tuberculosis in E. coli. The gene coding Rv3369 protein was amplified by polymerase chain reaction (PCR), then was inserted into an expression vector pET28a to get recombinant plasmid. The recombinant plasmid was transformed into E. coli BL21(DE3) and induced by IPTG. The expressed product was indentified by SDS-PAGE and purified by Ni- NTA His. Bind Resin. The sequence of Rv3369 in recombinant plasmid was the same with GenBank's report. The molecular mass of the product is 19.5kDa, which accounts for about 20% in the thalli proteins, and its purity is more than 90% analyzed by SDS-PAGE and laser scanning. The yield of recombinant protein is 1.56mg from 100mL of culture. Compared with other methods, purity of the recombinant protein is higher through affinity chromatography.
Insights
Researchers successfully produced high-purity recombinant Rv3369 protein from Mycobacterium tuberculosis using E. coli expression. This method yielded 1.56mg of protein with over 90% purity, demonstrating its effectiveness for Mycobacterium tuberculosis research.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Mycobacterium tuberculosis Rv3369 protein is a potential target for therapeutic intervention.
- Efficient expression and purification of recombinant Rv3369 are crucial for its functional and structural studies.
- Current methods for protein purification may lack the required purity and yield for detailed analysis.
Purpose of the Study:
- To develop a method for obtaining purified recombinant Rv3369 protein from Mycobacterium tuberculosis.
- To optimize the expression and purification process of Rv3369 in E. coli.
- To characterize the purity and yield of the expressed recombinant Rv3369 protein.
Main Methods:
- Amplification of the Rv3369 gene using polymerase chain reaction (PCR).
- Cloning the gene into the pET28a expression vector to create a recombinant plasmid.
- Transformation of the recombinant plasmid into E. coli BL21(DE3) and induction with IPTG.
- Purification of the expressed protein using Ni-NTA His.Bind Resin and analysis by SDS-PAGE.
Main Results:
- The sequence of the Rv3369 gene in the recombinant plasmid matched GenBank records.
- The expressed recombinant Rv3369 protein has a molecular mass of 19.5kDa.
- The protein constituted approximately 20% of the total cellular proteins.
- SDS-PAGE and laser scanning analysis confirmed a purity exceeding 90%.
- A yield of 1.56mg of recombinant protein was obtained from 100mL of bacterial culture.
Conclusions:
- The expression of recombinant Rv3369 protein in E. coli is feasible and efficient.
- Affinity chromatography using Ni-NTA His.Bind Resin provides a high-purity method for Rv3369 purification.
- This optimized method yields a high-purity recombinant Rv3369 protein suitable for further research.
- The developed protocol offers an improved approach compared to other purification techniques.
