Hitrap heparin column

The full-length rnt, sbcb, and exox genes were amplified by PCR using E. coli genomic DNA from JM109 or K-12 strains and cloned into NdeI/XhoI sites of expression vectors pET-28a (Novagen) to generate the N-terminal His-tagged fused recombinant proteins. The expression plasmid was transformed into the E. coli BL21-CodonPlus(DE3)-RIPL strain (Stratagene) cultured in LB medium supplemented with 35 µg/ml kanamycin. Cells were grown to an OD₆₀₀ of 0.5–0.6 at 37°C and induced by 0.8 mM IPTG at 18°C for 18 h. The harvested cells were dissolved in 50 mM Tris-HCl (pH 7.5) buffer containing 300 mM NaCl and disrupted by a microfluidizer. Each exonuclease was purified by chromatographic methods using a HiTrap TALON column (GE Healthcare), a HiTrap Heparin column (GE Healthcare), and a gel filtration column (Superdex 75, GE Healthcare). Purified RNase T, ExoI, and ExoX samples were concentrated to 15–35 mg/ml in 300 mM NaCl and 50 mM Tris-HCl (pH 7.0).

Human MSH2 and His-tagged MSH3 were overexpressed in SF9-insect cells using a pFastBac dual-expression system (GIBCO-BRL), and purified as follows11 (link)30 (link). MSH2 and his-tagged MSH3 were overexpressed in SF9-insect cells using a pFastBac dual-expression system. Briefly, the supernatant was loaded onto a 5 ml HiTrap chelating column (GE Healthcare) charged with Ni-NTA affinity column and equilibrated with lysis buffer. The bound proteins were then eluted with a 25 ml 20–200 mM imidazole gradient. The peak fractions containing the MSH2/MSH3 (eluted at 140 mM imidazole) and were then loaded onto a Mono P and HiTrap Heparin column (GE Healthcare) connected in tandem and equilibrated in column buffer (25 mM HEPES NaOH, pH 8.1, 0.1 mM EDTA, 10% glycerol (v/v) and 1 mM DTT) containing 300 mM NaCl. The MSH2/MSH3 containing fractions were then applied to MonoQ (GE Healthcare). MSH2/MSH3 fractions were stored in 20% glycerol (v/v), aliquoted and frozen at −80 °C.

For the human Rab1a construct (plasmid pJB78 GST-TEV-Rab1a(1–204)-Q70L-C26S-C126S) that was used for maleimide labelling, a WT Rab1a (1–204) fragment was mutated into Q70L also the surface-exposed cysteines were mutated to serines (C26S-C126S) so that only one Cys was left at the C terminus of the protein. Protein was overexpressed in E. coli C41(DE3)RIPL, purified on Glutathione Sepharose 4B resin (GE Healthcare 17-0756-05), followed by the removal of the GST tag with TEV protease overnight. The cleaved protein was diluted to a final concentration of 50–100 mM NaCl in dilution buffer (20 mM HEPES 8.0 and 1 mM TCEP) and passed through a 5 mL HiTrap Q column (GE Healthcare, 17505301) followed by a 5 mL HiTrap Heparin column (GE Healthcare, 17040601) to remove cleaved GST and TEV protease. The heparin flow-through was concentrated, loaded with nucleotide and MgCl₂, purified and concentrated in the same way as Rab5a.
For HXD-MS, a Rab1a–Q70L construct was designed with last 2 residues (CC) deleted. This construct (plasmid pYO1262 His-TEV-Rab1a(1-203)-Q70L) was expressed and purified by affinity chromatography on Ni-NTA, ion-exchange chromatography on Q column and Heparin column and gel filtration on Superdex 75 16/60.

Insect cells were infected with the 6His-Skp1 baculovirus, and 2×10⁹ cells were harvested from 1 L culture after 72 h. The cells were resuspended in R buffer (20 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1 mM dithiothreitol [DTT], 10% glycerol) containing 300 mM NaCl and homogenized, and the sample was centrifuged at 30,000× g for 30 min. Clarified lysate was directly loaded onto TALON Metal Affinity Resin (Clontech). Bead-bound proteins were eluted in R buffer containing 300 mM NaCl and 300 mM imidazole and lacking DTT and EDTA. The sample was diluted 3-fold with R buffer and loaded onto a HiTrap Heparin column (GE Healthcare). The pass-through fraction was loaded onto a HiTrap Q column (GE Healthcare), and 6His-Skp1 was eluted at ∼550 mM NaCl with a linear gradient of 0.1–1.0 M NaCl in R buffer. Peak fractions were collected and dialyzed against T buffer (20 mM Tris-HCl [pH 7.5], 50% glycerol, 1 mM DTT) containing 300 mM NaCl. The Protein Assay Kit (Bio-Rad), with bovine serum albumin as the standard protein, was used for determination of the protein concentration of Skp1.

A 2 L of IPTG-induced cells containing RM-intein-CBD or 6 L of IPTG-induced cells of S-intein-CBD were resuspended in 60 ml and 180 ml of chitin column buffer and the suspensions sonicated to lyse the cells. After the removal of the cell debris by the centrifugation at 15,000 rpm at 4°C for 30 min, clarified cell lysates were loaded onto 20-ml chitin columns by gravity flow (1 column for RM-intein-CBD, 2 columns for S-intein-CBD). The CBD-tagged enzymes in flow-through were reloaded 3 times and the columns were washed with 10 column volumes of chitin buffer (∼200 ml). A 20 ml of cleavage buffer (chitin column buffer + 50 mM DTT) was added to the top of chitin column. Approximately 2 ml of cleavage buffer passed through the column and the flow-through was discarded. The DTT-catalyzed intein cleavage reaction continued at 4°C for 2-3 days. The eluted proteins from chitin columns were diluted in a low salt buffer to reach 0.1-M NaCl concentration, which was subsequently loaded onto 5-ml Hi-Trap Heparin column (GE Healthcare). After the extensive washing with low salt, the RM or S protein was eluted with a salt gradient (0.1–1-M NaCl). Eluted peak fractions were analyzed by SDS-PAGE, concentrated in protein concentrators and protein was resuspended in enzyme storage buffer (0.2 M NaCl, 20 mM Tris-HCl, pH 7.5, 10 mM DTT, 0.5 mM EDTA, 50% sterile glycerol) to be stored at –20°C.