Improved mycobacterial protein production using a Mycobacterium smegmatis groEL1ΔCexpression strain

Abstract

BackgroundThe non-pathogenic bacterium Mycobacterium smegmatis is widely used as a near-native expression host for the purification of Mycobacterium tuberculosis proteins. Unfortunately, the Hsp60 chaperone GroEL1, which is relatively highly expressed, is often co-purified with polyhistidine-tagged recombinant proteins as a major contaminant when using this expression system. This is likely due to a histidine-rich C-terminus in GroEL1.ResultsIn order to improve purification efficiency and yield of polyhistidine-tagged mycobacterial target proteins, we created a mutant version of GroEL1 by removing the coding sequence for the histidine-rich C-terminus, termed GroEL1ΔC. GroEL1ΔC, which is a functional protein, is no longer able to bind nickel affinity beads. Using a selection of challenging test proteins, we show that GroEL1ΔC is no longer present in protein samples purified from the groEL1ΔC expression strain and demonstrate the feasibility and advantages of purifying and characterising proteins produced using this strain.ConclusionsThis novel Mycobacterium smegmatis expression strain allows efficient expression and purification of mycobacterial proteins while concomitantly removing the troublesome contaminant GroEL1 and consequently increasing the speed and efficiency of protein purification.

Mycobacterium tuberculosis Maltosyltransferase GlgE, a Genetically Validated Antituberculosis Target, Is Negatively Regulated by Ser/Thr Phosphorylation

“…4B). We also noticed that the M. smegmatis GroEL1 protein was co-purified in each preparation, presumably due to the presence of a known His-rich region capable of binding to the nickel-bearing beads, as reported earlier (34). This inadvertently provided an internal FIGURE 4.…”

Systematic Analysis of Mycobacterial Acylation Reveals First Example of Acylation-mediated Regulation of Enzyme Activity of a Bacterial Phosphatase

“…The M. smegmatis 60-kDa chaperone (GroEL, MSMEG_1583) was found as an additional band in most of the protein purifications. Because of the presence of a C-terminal histidine-rich region, GroEL tends to copurify with the overexpressed His-tagged proteins (64,65). We checked the enzyme activities of three proteins to show that the proteins purified from M. smegmatis are properly folded and functionally active.…”

Mycobacterium tuberculosis Prokaryotic Ubiquitin-like Protein-deconjugating Enzyme Is an Unusual Aspartate Amidase

“…Dop and PupϳIno1 Purification-Dop-His 6 and PupϳIno1-His 6 were purified essentially as described previously (8), except we used a M. smegmatis strain with a C-terminal deletion in GroEL1 (15). This deletion removes a polyhistidine sequence in GroEL1, which eliminates co-purification of GroEL1 with target proteins.…”