title: Improved mycobacterial protein production using a Mycobacterium smegmatis groEL1ΔC expression strain.
abstract: BACKGROUND
  The 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  This 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.
authors: European Molecular Biology Laboratory (EMBL), Hamburg Outstation, c/o DESY, Building 25a, Notkestrasse 85, 22603 Hamburg, Germany. e.noens@embl-hamburg.de
authors: Elke E
authors: EE
authors: Noens
authors: Chris
authors: C
authors: Williams
authors: Madhankumar
authors: M
authors: Anandhakrishnan
authors: Christian
authors: C
authors: Poulsen
authors: Matthias T
authors: MT
authors: Ehebauer
authors: Matthias
authors: M
authors: Wilmanns
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018441:Biofilms / Q000254:growth & development; D015139:Blotting, Southern; D018834:Chaperonin 60 / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005818:Genetic Engineering / Q000379:methods; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D004269:DNA, Bacterial; D018832:Molecular Chaperones