Chelating sepharose fast flow

The E6E7 recombinant protein was expressed in the BL21(DE3) Star pLysS (Novagen) E. coli strain. Isolated colonies grown on LB agar were transferred into 0.5 L of LB with ampicillin and incubated in a shaker at 37°C until the optical density at 600 nm (OD₆₀₀) reached 0.6. Protein expression was induced by adding 1 mM isopropyl-β-thiogalactopyranoside and the cultures were incubated at 37°C for 4 h. Cells were harvested by centrifugation and lysed using a French press. Bacterial inclusion bodies were washed in washing buffer (PBS pH 7.5, 250 mM NaCl and 2 M urea) and solubilized with binding buffer (PBS pH 7.5, 250 mM NaCl, 8 M urea, 5 mM β-mercaptoethanol and 5 mM imidazole).
The recombinant protein was purified on Chelating-Sepharose^™ Fast Flow (GE Healthcare). The columns with adsorbed protein were washed three times with binding buffer containing 60 mM, 80 mM and 100 mM imidazole. The E6E7 protein was eluted with elution buffer (PBS pH 7.5, 250 mM imidazole and 8 M urea). Stepwise dialysis was performed until urea reached a concentration of 2 M. The protein was quantified by spectrophotometry (NanoDrop, Thermo Scientific Inc.) according to its molar extinction coefficient.
The recombinant protein was characterized by SDS-PAGE and mass spectrometry (S1 Material and Methods).

To prepare an expression vector for His-CiAPEX2, a CiAPEX2 cDNA fragment containing 6 histidine at its N-terminus was PCR amplified from the pGEX-4T-1-CiAPEX2 vector using PCR primers 5′-GCATCCCATGGAACATCATCATCATCATCATATGAAAATACTAACATGGAACATC-3′ and 5′-CCGGAATTCTCATTTCTTTTTGTCCCATTC-3′. The amplified fragment containing NcoI and EcoRI site at its 5′ and 3′ ends, respectively, was inserted into a pTrc99A vector. The procedure for expression and supernatant preparation was the same as GST-CiAPEX2 purification with 2 exceptions; incubation temperature after IPTG induction is 16 °C and using buffer D (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol) containing 10 mM imidazole instead of buffer A as a resuspending buffer. The supernatant was applied to a Chelating Sepharose Fast Flow (GE Healthcare) column and the His-CiAPEX2 was eluted from the column with buffer D containing 500 mM imidazole. After dialysis against buffer E (30 mM Tris-HCl pH 7.5, 135 mM NaCl, 2 mM EDTA), fraction containing His-CiAPEX2 was loaded onto HiTrap-Q column (GE Healthcare), and eluted in Tris buffer (50 mM Tris-HCl pH 7.5, 2 mM EDTA) containing 0.235-0.32 M NaCl. The purified His-CiAPEX2 protein was dialysis against buffer C and stored at −80 °C until use.

The resin for protein purification, the Chelating Sepharose Fast Flow, was obtained from GE (Uppsala, Sweden). The standard of DFA III was purchased from Wako Pure Chemical Industries (Osaka, Japan). Electrophoresis reagents were purchased from Bio-Rad (Hercules, CA, USA). Isopropyl-β-D-1-thiogalactopyranoside (IPTG) and all chemicals used for enzyme assays and characterization were at least of analytical grade obtained from Sigma (St. Louis, MO, USA) or Sinopharm Chemical Reagent (Shanghai, China).

Escherichia coli BL21 (DE3, Invitrogen) was transformed with pet16b-UBwt (1-76, human) and grown in LB medium containing 100 μg/ml ampicillin at 37 °C. Protein expression was induced with 1 mM IPTG at OD₆₀₀ of 0.7-0.9 and cultured for additional 6 h at 37 °C. Cells were harvested by centrifugation and frozen at -20 °C. Ubiquitin purification was carried out according to optimized protocol (Beers and Callis, 1993 (link)). In brief, cells from 4 liters culture were resuspended in 200 ml of lysis buffer (50 mM NaH2PO4 pH=8.0, 300 mM NaCl, 1 mM imidazole) containing 1 mg/ml lysozyme. After incubation of cells on ice (10 min), NaCl and PMSF were added to final concentrations 600 mM and 2 mM, respectively. Cells were raptured by sonication. Cleared supernatant was loaded on a Chelating Sepharose Fast Flow (GE Healthcare) charged with nickel ions. The column was subsequently washed with lysis buffer, lysis buffer without NaCl, lysis buffer adjusted to pH=5.5 and pH=4.5. Protein was eluted with lysis buffer supplemented with 250 mM imidazole.
As the last purification step size exclusion chromatography on HiLoad 16/60 Superdex 75 prep grade (GE Healthcare) equilibrated with PBS pH=7.4 was used. Ubiquitin was stored for further experiments at 0.5-2 mM concentration at -20 °C.

V2.24 fHbp was amplified from N. meningitidis OX99.32412 and SNPs introduced by PCR, then ligated into pET21b using Quick-Stick Ligase (Bioline). Versions of fHbp were ligated into pET28a-His-MBP-TEV (in frame with sequence encoding a histidine tag and the Escherichia coli maltose-binding protein (MBP) with a C-terminal TEV cleavage site) linearised with XhoI, and constructs confirmed by sequencing.
v2.24 fHbps were expressed in E. coli B834 during growth at 22°C for 24 hrs with 1 mM IPTG (final concentration). Bacteria were harvested and resuspended in Buffer A (50 mM Na-phosphate pH 8.0, 300 mM NaCl, 30 mM imidazole) and the fHbp purified by Nickel affinity chromatography (Chelating Sepharose Fast Flow; GE Healthcare). Columns were washed with Buffer A, then with 80:20 Buffer A:Buffer B (50 mM Na-phosphate pH 8.0, 300 mM NaCl, 300 mM Imidazole), and proteins eluted in 40:60 Buffer A:Buffer B. Proteins were dialysed overnight at 4°C into PBS, 1mM DTT pH 8.0 with TEV protease prior to Nickel affinity chromatography to remove the HIS-GST-TEV. fHbp was eluted from Sepharose columns with Buffer B after washing with buffer C (50 mM Na-phosphate pH 6.0, 500 mM NaCl, 30 mM Imidazole), and dialysed overnight at 4°C into Tris pH 8.0. fHbp v1.1^I311A expression and purification was performed as described previously [83 (link)].

To prepare polyclonal anti-Lrp antibodies, Lrp was produced from the recombinant strain KA014 that expressed Lrp-his₆ after induction at 30 °C for 6 h with 0.1 mM IPTG, and the proteins in cell lysate were fractionated by gel electrophoresis. The gel containing Lrp was excised, grounded, and mixed with complete (first injection) or incomplete adjuvant (other injections) for immunization of the 6–8 weeks old BALB/c mice by intraperitoneal injection. Mouse serum was collected after five consecutive injections every week, and stored at −80 °C. In Western blot hybridization, the proteins in total cell lysate were transferred onto a PVDF membrane after fractionation by SDS-polyacrylamide gel electrophoresis. The target proteins on the membrane were hybridized with relevant primary antibodies followed by secondary antibodies conjugated to horseradish peroxidase (HRP), and visualized by enhanced chemiluminescence. To prepare the anti-MARTX antibodies, the C-terminal his₆-tagged ERM domain of V. vulnificus MARTX produced in E. coli NovaBlue (DE3) was purified by Chelating Sepharose Fast Flow (GE Healthcare). Purified ERM peptide was then used to generate polyclonal rabbit anti-ERM antiserum (AngeneBiotech, Taipei).

PslG_31‐442 was expressed and purified as previously described (Yu et al., 2015). The first 30 residues of PslG were truncated because they were predicted to be a signal peptide by the Signal P4.1 server. Briefly, E. coli BL21 (DE3) carried pGLO1‐pslG was grown in 1 L LB containing 100 μg/ml ampicillin at 37°C. When the OD₆₀₀ of the culture reached 0.5–0.8, protein expression was induced overnight with 0.1 mM isopropyl β‐D‐thiogalactopyranoside at 22 ºC. Bacteria cells were harvest by centrifugation at 4,000 rpm for 30 min at 4 ºC and resuspended in buffer A (25 mM Tris‐HCl, pH 8.0, 200 mM NaCl, 60 mM imidazole). The bacterial suspension was lysed by sonication and centrifuged at 16,000 rpm for 30 min at 4 ºC. The supernatant was applied to a nickel affinity column (Chelating Sepharose Fast Flow, GE Healthcare), and washed with three column volumes of binding buffer to remove the non‐specific proteins. The expressed protein was eluted with buffer B (25 mM Tris‐HCl, pH 8.0, 200 mM NaCl, 250 mM imidazole). The eluted fraction containing the protein was purified by size‐exclusion chromatography (Superdex 200 10/300 GL, GE Healthcare) with buffer C (10 mM Tris‐HCl, pH 8.0, 100 mM NaCl, 5% (v/v) glycerol). The purified PslD was a gift from prof. Lichuan Gu.