Improved mycobacterial protein production using a Mycobacterium smegmatis groEL1ΔCexpression strain
© Noens et al; licensee BioMed Central Ltd. 2011
Received: 17 December 2010
Accepted: 25 March 2011
Published: 25 March 2011
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.
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.
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.
Heterologous expression of recombinant proteins in Escherichia coli can result in the production of insoluble inclusion bodies. Recent statistics show that less than half of the M. tuberculosis (Mtb) proteins expressed in E. coli are soluble . Therefore, the non-pathogenic bacterium Mycobacterium smegmatis is often used as an alternative, more closely related host for the expression of mycobacterial proteins. Furthermore, M. smegmatis may also provide mycobacterium-specific chaperones, which can help correct folding of Mtb proteins .
During nickel affinity purification, it has been observed that a protein of 56 kDa is co-purified with polyhistidine-tagged recombinant proteins while using M. smegmatis as an expression system. This contaminant was previously identified as the Hsp60 chaperone GroEL1 of M. smegmatis [1–3]. The protein sequence of GroEL1 shows a histidine-rich C-terminus (7 out of 11 amino acids are histidines), which is likely to be the reason for the observed nickel sepharose binding [1, 2].
Unlike most other bacteria, mycobacteria possess two Hsp60 chaperone groEL genes, one of which is arranged in the bicistronic groESL operon . M. smegmatis also encodes a third Hsp60 protein (Msmeg1978), which is more distantly related to GroEL1 (Msmeg1583) and GroEL2 (Msmeg0880) . Although groEL1 of M. smegmatis can be found in the same operon as groES, an arrangement indispensable for the chaperone function in bacteria, its histidine-rich tail is distinct from the more typical glycine-methionine-rich C-terminal region found in GroEL2 . Furthermore, groEL2 is an essential gene and exists in all actinobacteria, in contrast to groEL1 [3, 5]. Recently, it has been shown that groEL2 and groES are expressed more strongly than groEL1, which might have arisen from a difference in stability of the predicted post-transcriptionally cleaved mRNAs for groES and groEL1 . Consistent with the current chaperone model in mycobacteria, one chaperone, here GroEL2, would act as the main house keeping chaperone in M. smegmatis, with the other chaperones (GroEL1 and Msmeg1978) adopting more specialised functions. Indeed, GroEL1 of M. tuberculosis was recently identified as being associated with nucleotides, suggesting a role as a DNA chaperone, while GroEL1 of M. smegmatis was found to have a role in mycolic acid biosynthesis during biofilm formation [5, 6, 3].
The co-purification of GroEL1 with histidine-tagged recombinant proteins can be particularly problematic since native GroEL1 is expressed at relatively high levels, meaning that in the case of a low yield of recombinant protein, GroEL1 may well compete with the protein of interest for binding sites on nickel affinity beads. Minimal sample manipulation is recommended during protein purification to improve efficiency. Therefore, additional steps required to remove GroEL1 can result in a significant loss of the protein of interest.
In this article, we describe an M. smegmatis expression strain containing a mutant version of GroEL1, termed GroEL1ΔC, which consists of a groEL1 gene without a coding sequence for the histidine-rich C-terminal tail. We show that GroEL1ΔC is a functional protein, which no longer co-purifies when using nickel affinity purification and we provide evidence that proteins purified from this strain are correctly folded, active and that they behave identically to those purified from the original expression strain. Taken together, our data demonstrate that M. smegmatis groEL1ΔC is a competent protein expression strain, which allows the efficient removal of the troublesome contaminant GroEL1 without the requirement of additional purification steps.
Bacterial strains and media
The E. coli strains DH5α (Invitrogen) and HB101 (Promega) were used for cloning of expression constructs and the target substrate to generate the mutant version of groEL1 using standard procedures . Transformants were selected in Luria Broth containing the appropriate antibiotics.
M. smegmatis mc2155 was used as the parent (wild type) strain for the groEL1ΔC strain. Both M. smegmatis strains were maintained in Middlebrook 7H9 or 7H10 medium supplemented with 0.2% (v/v) glycerol, 10% ADC, 0.05% (v/v) tween-80 and the appropriate antibiotics.
For the expression of the recombination proteins in M. smegmatis in order to create the mutant form of groEL1, 0.2% succinate (w/v) was added as a carbon source to 7H9 medium supplemented with 0.2% (v/v) glycerol, 0.05% (v/v) tween and the appropriate antibiotics. Expression of his-tagged recombinant proteins in M. smegmatis was performed in 7H9 medium supplemented with 0.2% (w/v) glucose as carbon source. Acetamide was added to a final concentration of 0.2% (w/v) at 0.5 OD600 and at 2.5 OD600 for the expression of the recombination proteins and his-tagged recombinant proteins, respectively.
Plasmids, constructs and oligonucleotides
Plasmids and constructs used in this study
Che9c recombination proteins under control of the acetamidase promoter in pLAM12
HygR cassette flanked by γδ-res sites and 2 MCSs
Expressing an γδ resolvase and tetracycline resistant
pYUB854 with a 520 bp fragment harbouring groEL1 (+1067/+1587, relative to groEL1) inserted upstream of the HygR cassette and a 555 bp fragment downstream of groEL1 including the STOP codon of groEL1, inserted downstream of the HygR cassette
Mycobacterial overexpression vector
Geerlof et al., unpublished data
Rv2109-2110 in pMYNT, Rv2110 is N-terminally his-tagged
Rv3280-3281 in pMYNT. Only his-tagged Rv3280 seems to express using this construct.
Rv3285 in pMyNT
Rv3874-3875 in pMYNT, Rv3874 is N-terminally his-tagged
Rv2523 in pMYNT
Primers used in this study
For the expression of M. tuberculosis proteins in M. smegmatis, the pMyNT expression vector was used [Geerlof et al., unpublished data]. pMyNT/ACPS, pMyNT/AccA3 and pMyNT/AccD5 were made as follows: PCR was performed with primer pair Rv2523-F & Rv2523-R for ACPS, accA3-F & accA3-R for AccA3 and accD5E5-F & accD5E5-R for AccD5 and the resulting fragments were digested with NcoI-HindIII and inserted into NcoI-HindIII digested pMyNT.
Creation of the groEL1ΔCmutant
The groEL1ΔC mutant was created using the mycobacterial recombineering method . pEN15 was digested with AflII and SpeI to create the linear target substrate, which was introduced into mc2155 electrocompetent cells, expressing the recombinase genes on pJV53 and in this way creating hygromycin-resistant transformants. The hygromycin-resistance cassette was removed using δγ resolvase, expressed on pGH542, generating an unmarked deletion .
Southern blot analysis
Genomic DNA (5ug) was isolated as described , digested with the appropriate enzymes, separated on a 0.9% agarose gel and transferred to a positively charged nylon membrane (Roche). For DNA probe labelling, hybridisation and detection, the DIG high prime DNA labelling and detection starter kit 1 (Roche) was used.
Bacterial growth was followed by measuring the optical densities at a wavelength of 600 nm as a function of time. Cultures were prepared with 7H9 expression medium (0.2% (w/v) glucose as carbon source) in identical triplicates for each strain. Duplicate samples were taken every 4 hours for 40 hours. When the optical density at 600 nm exceeded 1.5, samples were diluted in order to remain within the linear range of the detector.
Protein expression and purification
All methods related to protein expression in M. smegmatis were carried out as described [12, 13]. Protein-protein complexes from operon-encoded proteins were expressed using the native operon structure . In brief, pellets from 500 ml cultures were dissolved in 30 ml lysis buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.5 M urea with protease inhibitor cocktail (Sigma) and 1 mg/ml DNase I (Serva). Resuspended cells were sonicated four times, each for 5 min (with a 0.3 s pulse and 0.7 s rest) at 5 min intervals to prevent overheating, using a Bandelin VW3200 probe at 45% amplitude. The supernatant was collected after centrifugation (30,000 × g) for 1 h at 4°C, filtered through a 0.44 μm filter and loaded onto a nickel affinity sepharose (NiAC) column. After washing with 10 column volumes of 50 mM Tris-HCl pH 8.0, 300 mM NaCl and 20 mM imidazole, proteins were eluted in 50 mM Tris-HCl, 100-150 mM NaCl and 250-500 mM imidazole and subjected to size exclusion chromatography using either a Superdex 75 (16/60) column (GE Healthcare) or, for large protein complexes, a Superose 6 (10/300) (GE Healthcare) with 25 mM Tris-HCl pH 8.0, 150 mM NaCl and 1 mM DTT as buffer. The collected protein samples were analysed by SDS-PAGE and concentrated accordingly.
Circular Dichroism (CD) spectrum analysis
CD measurements were performed on a Jasco J-810 spectropolarimeter. Prior to measurement, samples were dialysed into 10 mM potassium phosphate, 150 mM NaCl, pH 7.4. Spectra were recorded between 182 and 260 nm in a 2 mm cuvette with machine settings as follows: 1 nm bandwidth, 1 sec response, 1 nm data pitch, 100 nm/min scan speed, cell length of 0.1 cm. Each curve presented is the average of three separate measurements.
Coupled enzyme assay
Enzymatic activity of the AccD5-AccA3 complex was estimated by a coupled enzyme assay that follows the rate of ATP hydrolysis spectrophotometrically . The production of ADP during the reaction was coupled to pyruvate kinase and lactate dehydrogenase, and the oxidation of NADH was probed at 340 nm. The assay mixture contained 7 units of pyruvate kinase, 10 units of lactate dehydrogenase, 50 mM NaHCO3, 3 mM ATP, 0.5 mM phosphoenol pyruvate, 0.2 mM NADH, 0.3 mg/ml BSA, 100 mM K2HPO4 pH 7.6 and 5 mM MgCl2 and varying concentrations of propionyl-coenzyme A. Reactions were initiated by the addition of enzyme to the assay mixture and were maintained at 30°C. Data were acquired using a Tecan infinite M1000 microplate reader. The kinetic parameters Km and Vmax were determined by fitting the mean velocities versus the substrate concentration to the Michaelis-Menten equation of enzyme kinetics using nonlinear regression analysis, executed by the program Prism 5 (GraphPad Software™).
Results and Discussion
Creation of the groEL1ΔCstrain
GroEL1ΔC is absent during nickel affinity purification of proteins expressed in M. smegmatis groEL1ΔC
List of test proteins used to validate the groEL1ΔC expression strain
Mol. Mass (kDa)
α- and β-subunit of the mycobacterial proteasome (α7β7β7α7 subunit organisation)
Using native operon content, producing a 730 kDa multimeric complex
α- and β-subunit from acyl-CoA carboxylase AccD5-AccA3 complex (α3β3β3α3 subunit organisation)
As monomeric proteins, mixed to form a acyl-CoA carboxylase complex of 740 kDa
Potential virulence factor CFP10-ESAT6 complex
Using native operon content, producing a heterodimeric (1:1) complex
Holo-acyl-carrier protein synthase
As monomeric protein
Proteins purified from M. smegmatis groEL1ΔCbehave identically to those purified from the wild type strain
M. smegmatis encodes three forms of the Hsp60 chaperone GroEL: Msmeg1583 (GroEL1), Msmeg0880 (GroEL2) and Msmeg1978. However, the precise molecular function of each protein remains unclear. Changing the last 18 amino acids of GroEL1 does not alter growth but does result in a strong defect in biofilm formation . To confirm that the newly created recombinant version of GroEL1 has no effect on the correct folding and, ultimately, the function of the proteins expressed in M. smegmatis groEL1ΔC, a number of different proteins and protein complexes have been expressed and analysed.
In the previous section, we have shown that it is possible to express and purify potentially challenging protein complexes, such as the proteasome complex PrcA-B and the CFP10-ESAT6 complex, from the recombinant groEL1ΔC strain. These data imply that the proteins isolated from the groEL1ΔC strain are correctly folded, since we were able to observe all components after purification. In both examples, complex formation requires direct protein-protein interactions between subunits of the complex as only one subunit is his-tagged.
Taking our analysis one step further, we directly tested the structural and functional properties of proteins isolated from the groEL1ΔC strain. We used the five expression constructs described above and transformed them into both M. smegmatis mc2155 and groEL1ΔC. Proteins were expressed and purified using a nickel affinity column as described above. AccD5 and AccA3 protein samples were mixed in a 1:1 stoichiometry to form the high-molecular-weight AccD5-AccA3 complex. Size exclusion chromatography was performed on all samples as a final purification step.
We have developed an M. smegmatis expression strain that allows efficient expression and purification of mycobacterial proteins, multi-subunit protein complexes and post-translationally modified proteins while concomitantly removing the troublesome contaminant GroEL1 and consequently increasing the speed and efficiency of protein purification. The M. smegmatis groEL1ΔC strain is particularly suitable for laboratories performing in vitro activity assays and structural studies on mycobacterial proteins and protein complexes.
Polymerase chain reaction
Heat shock protein 60
Nickel affinity sepharose column
sodium dodecyl sulfate polyacrylamide gel electroporesis
matrix-assisted laser desorption/ioization reflection time-of-flight.
Acknowledgements and Funding
We thank Arie Geerlof for the pMyNT expression vector, Young-Hwa Song for her contribution in the early stages of the work, the Proteomics Core Facility of EMBL Heidelberg for performing peptide mass fingerprinting, the Mandelkow Lab (Max Planck Institute for Structural Molecular Biology, Hamburg, Germany) for access to their circular dichroism spectroscope. CW is funded by a Rubicon post-doctoral fellowship (825.08.023) from the Netherlands organization for scientific research (NWO). ME is funded by an EMBO long-term fellowship (ALTF-7272008). The project has been supported by grants awarded to MW from BMBF (Pathogenomik Plus PTJ-BIO 0313801L), from the European Commission Framework VII (NATT, 222965 and SystemTB, 241587) and from the DFG (SPP1170, WI 1058/6-3).
- Goldstone RM, Moreland NJ, Bashiri G, Baker EN, Lott JS: A new Gateway® vector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis. Protein Expr Purif. 2008, 57: 81-87. 10.1016/j.pep.2007.08.015.View ArticleGoogle Scholar
- Poulsen C, Ahkter Y, Jeon AH, Schmitt-Ulms G, Meyer HE, Stühler K, Wilmanns M, Song YH: Proteome-wide identification of mycobacterial pupylation targets. Mol Syst Biol. 2010, 6: 386-394. 10.1038/msb.2010.39.View ArticleGoogle Scholar
- Ojha A, Anand M, Bhatt A, Kremer L, Jacobs WR, Hatfull GF: GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in Mycobacteria. Cell. 2005, 123: 861-873. 10.1016/j.cell.2005.09.012.View ArticleGoogle Scholar
- Rinke de Wit TF, Bekelie S, Osland A, Miko TL, Hermans PW, van Soolingen D, Drijfhout JW, Schöningh R, Janson AA, Thole JE: Mycobacteria contain two groEL genes: the second Mycobacterium leprae groEL gene is arranged in an operon with groES. Mol Microbiol. 1992, 6 (14): 1995-2007. 10.1111/j.1365-2958.1992.tb01372.x.View ArticleGoogle Scholar
- Rao T, Lund PA: Differential expression of the multiple chaperonins of Mycobacterium smegmatis. FEMS Micobiol Lett. 2010, 310: 24-31. 10.1111/j.1574-6968.2010.02039.x.View ArticleGoogle Scholar
- Basu D, Khare G, Singh S, Tyagi A, Khosla S, Mande SC: A novel nucleoid-associated protein of Mycobacterium tuberculosis is a sequence homolog of GroEL. Nucleic Acids Res. 2009, 37 (15): 4944-4954. 10.1093/nar/gkp502.View ArticleGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: a laboratory manual. 1989, Cold Spring harbor: Cold Spring harbor laboratory pressGoogle Scholar
- Recht J, Kolter R: Glycopeptidolipid acetylation affects sliding motility and biofilm formation in Mycobacterium smegmatis. J Bacteriol. 2001, 183: 5718-5724. 10.1128/JB.183.19.5718-5724.2001.View ArticleGoogle Scholar
- van Kessel JC, Hatfull GF: Recombineering in Mycobacterium tuberculosis. Nat Methods. 2007, 4 (2): 147-152. 10.1038/nmeth996.View ArticleGoogle Scholar
- Bardarov S, Bardarov S, Pavelka Ms, Sambandamurthy V, Larsen M, Tufariello J, Chan J, Hatfull G, Jacobs WR: Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology. 2002, 148: 3007-3017.View ArticleGoogle Scholar
- Piuri M, Hatfull GF: A peptidoglycan hydrolase motif within the mycobacteriophage TM4 tape measure protein promotes efficient infection of stationary phase cells. Mol Microbiology. 2006, 62: 1569-1585. 10.1111/j.1365-2958.2006.05473.x.View ArticleGoogle Scholar
- Poulsen C, Holton S, Geerlof A, Wilmanns M, Song YH: Stoichiometric protein complex formation and over-expression using the prokaryotic native operon structure. FEBS Lett. 2010, 584: 669-674. 10.1016/j.febslet.2009.12.057.View ArticleGoogle Scholar
- Daugelat S, Kowall J, Matthow J, Bumann D, Winter R, Hurwitz R, Kaufmann SH: The RD1 proteins of Mycobacterium tuberculosis: expression in Mycobacterium smegmatis and biochemical characterization. Microbes Infect. 2003, 5: 1082-1095. 10.1016/S1286-4579(03)00205-3.View ArticleGoogle Scholar
- Diacovich L, Peiru S, Kurth D, Rodriguez E, Podesta F, Khosla C, Gramajo H: Kinetic and Structural Analysis of a New Group of Acyl-CoA Carboxylases Found in Streptomyces coelicolor A3(2). J Biol Chem. 2002, 277: 31228-31236. 10.1074/jbc.M203263200.View ArticleGoogle Scholar
- Xu Z, Horwich AL, Sigler PB: The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature. 1997, 388 (6644): 741-50. 10.1038/41944.View ArticleGoogle Scholar
- Fukami TA, Yohda M, Taguchi H, Yoshida M, Miki K: Crystal structure of chaperonin-60 from Paracoccus denitrificans. J Mol Biol. 2001, 312 (3): 501-9. 10.1006/jmbi.2001.4961.View ArticleGoogle Scholar
- Kumar CM, Khare G, Srikanth CV, Tyagi AK, Sardesai AA, Mande SC: Facilitated oligomerization of mycobacterial GroEL: Evidence for phosphorylation-mediated oligomerization. J Bacteriol. 2009, 191: 6525-6538. 10.1128/JB.00652-09.View ArticleGoogle Scholar
- Andersson SGE, Sharp PM: Codon usage in the Mycobacterium tuberculosis complex. Microbiology. 1996, 142: 915-925. 10.1099/00221287-142-4-915.View ArticleGoogle Scholar
- Gago G, Kurth D, Diacovich L, Tsai SC, Gramajo H: Biochemical and structural characterization of an essential acyl Coenzyme A carboxylase from Mycobacterium tuberculosis. J Bacteriol. 2006, 188: 477-486. 10.1128/JB.188.2.477-486.2006.View ArticleGoogle Scholar
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