Purification of Recombinant Mycobacterial Proteins

The purification of the recombinant proteins was carried out as described before (28 (link)). Briefly, the plasmids containing genes of interest fused to 6xHis-tags were transformed and expressed in E. coli BL21(DE3) strain and the recombinant proteins were later purified by metal ion affinity chromatography. The individual proteins PE18 (Rv1788), PE31 (Rv3478) and PPE26 (Rv1789) were purified by the on-column refolding method using an 8M Urea gradient. Purity was assessed by SDS-PAGE (>97% purity) and identity confirmed by Western blot. Molecular weight references for SDS-PAGE and Western blot were provided using PageRuler Unstained Protein Ladder (Thermofisher, 26614) and PageRuler Prestained Protein Ladder (Thermofisher, 26616), respectively. The concentration of the recombinant proteins was determined using the Lowry assay (BioRad Laboratories, Inc.) and the endotoxin content determined using Limulus amebocyte lysate (LAL) test (PYROTELL^®-T Lysate).

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García-Bengoa M., Vergara E.J., Tran A.C., Bossi L., Cooper A.M., Pearl J.E., Mussá T., von Köckritz-Blickwede M., Singh M, & Reljic R. (2023). Immunogenicity of PE18, PE31, and PPE26 proteins from Mycobacterium tuberculosis in humans and mice. Frontiers in Immunology, 14, 1307429.

Publication 2023

PageRuler Unstained Protein Ladder PageRuler Prestained Protein Ladder Lowry assay

Corresponding Organization : Lionex (Germany)

Other organizations : St George's, University of London, University of Leicester, Eduardo Mondlane University

Top 5 similar protocols

Variable analysis

independent variables

Plasmids containing genes of interest fused to 6xHis-tags

dependent variables

Purity of recombinant proteins
Identity of recombinant proteins
Concentration of recombinant proteins
Endotoxin content of recombinant proteins

control variables

Transformation and expression of recombinant proteins in E. coli BL21(DE3) strain
Purification of recombinant proteins by metal ion affinity chromatography
On-column refolding method using an 8M Urea gradient
SDS-PAGE and Western blot for assessment of purity and identity
Molecular weight references provided by protein ladders
Lowry assay for determination of protein concentration
Limulus amebocyte lysate (LAL) test for endotoxin content determination

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