Feng et al., Supplementals
Materials and Methods
Chemical cross-linking
Cross-linking with ethylene glycol bis-succinimidylsuccinate (EGS, Pierce) was conducted as described earlier (1) with minor changes.In the cross-linking reactions purified BplA or BirA protein (~1 μg/μl, 15 μl in total volume) were each mixed with cross-linker at different concentrations (0, 1.0, 5.0 and 20 μM). We also mixed together BplA and BirA at a molar ratio of 1:1 before the cross-linking reaction, to test the possibility that the proteins can form hybrid multimer species. All reactions were kept for 30 min at room temperature, and the r reaction products were separated by gradient SDS-PAGE (4-20%).
Protein expression and purification
The N-terminal hexahistidine-tagged F. novicida BplA and BirA proteinswere produced at high levels from pET28-bplA and pET28-birA using strain BL21(DE3) as the host (Table 1) whereas the hexahistidine-taggedE. coli BirA was obtained as described previously (2). When the optical density at wavelength of 600 nm (OD600 nm) reached ~ 0.8, bacterial cultures (~200 ml) were induced with 0.3 mM IPTG and kept at 30°C for ~3 h. The pelleted bacterial cells were re-suspended in ice-cold 1X PBS buffer (101.4 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 10% glycerol, pH7.4) containing 25 mM imidazole and lysed with a French pressure cell. The clarified supernatant was loaded onto a nickel chelate column (Qiagen). Following removal of contaminating proteins by washing the column with 10 column volumes of 1X PBS buffer containing 50mM imidazole, the proteins were eluted with 150 mM imidazole, dialyzed against PBS buffer and concentrated by ultra-filtration (30 kDa cut-off, Amicon Ultra) (1). Protein purity was assayedby gradient SDS-PAGE (4-20%).
Liquid chromatography quadrupole time-of-flight mass spectrometry
To determinethe identity of the recombinant BplA and BirAprotein, the de-stained SDS-PAGE gel slices were subjected to liquid chromatography quadrupole time-of-flight mass spectrometryusing a Waters Q-Tof API-US Quad-ToF mass spectrometer linked to a Waters nanoAcquity UPLC (3-5). Briefly, the gel slice of interest was digested with 25µl of Trypsin (G-Biosciences, St. Louis, MO) using a CEM Discover Microwave Digestor (Mathews, NC) for 15min at 55°C and 50W. The samples of the resultant peptides (~10µl) were loaded on a Waters Atlantis C-18 column (0.03mm particle, 0.075mm×150mm) and eluted at a flow rate of 250nl per min. Data acquisition was executed with the mass spectrometer and ms/ms analyses wereconducted on the most abundant four peaks at any given time.Data processing was performed using Waters Protein Lynx Global Server 2.2.5, Mascot (Matrix Sciences) combined with BLAST against NCBI nr database(6, 7)
Electrophoretic mobility shift assays
BplA and BirA were tested for the DNA binding activity by EMSA tests carried out by minor modifications of the prior procedure (4, 5). Double stranded DNA probes were generated by annealing two complementary oligonucleotides in 10 mM Tris-HCl cnotaining 1 mM EDTA and 100 mM NaCl (pH 8.0) and then labeled with DIG-ddUTP by terminal transferase(Roche).. The probes were the Ec bioO probe (52 bp) and the Fn bioO probe (51 bp) (Table S2). Following a 20 min of incubation of the DIG-labeled DNA probes (0.2 pmol) with/without BplA (or BirA) protein in binding buffer (Roche) at room temperature, the DNA-protein complexes formed were separated on 7% native PAGE gels and the chemi-luminescence signal was captured by the exposure of the nylon membrane to ECL film (Amersham) (8, 9).
Streptavidin blot analyses
Log-phase cultures (5 ml each) of F. novicida and E. coli were used to prepare crude extracts for streptavidin blot analyses. The extract of the wild type MG1655 strain of E coli K-12 was a positive control and the F. novicida extracts were from the ΔbioJ mutant (ΔFTN_0818) and two transposon mutant strains (the bplA mutant, FTN_0568 with the insertion at site 81, and birA mutant, FTN_0811 with the insertion at site 129). These crude extracts were made from equivalent amounts of cells and were loaded on 4-20% gradient SDS-PAGE gels (Bio-Rad). The proteins were transferred to a membrane (Immobilon-P from Millipore) by a semi-dry method. After transfer the membrane was rinsed with maleic acid buffer (100 mM maleic acid, 150 mM NaCl, pH 7.5), followed by 1 h incubation of the membrane with the blocking buffer (maleic acid buffer plus 1% blocking reagent) (Roche). The membrane was then incubated for 1 h with maleic acid buffer containing a streptavidin-AP-conjugate (0.02%), and then washed twice (15 min each) with washing buffer (maleic acid buffer containing 0.3% Tween 20). Following incubation (~10 min) of the membrane with CDP-Star (Roche) in detection buffer (100 mM Tris-HCl, 100 NaCl, pH 9.5), the membrane was exposed to Amersham HyperfilmECL high performance chemi-luminescence film.
Bioinformatic analyses
Alignments were done using ClustalW2 and the outputswere processed with the ESPript 2.2 server SPDBV_4.01 was used for structural modeling of the F. novicidaAccB protein.
Supplemental Materials and Methods References
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