ABCB10A (4AYW) Materials & Methods |
Entry Clone Source: MGC |
Entry Clone Accession: IMAGE: 6143235 |
SGC Construct ID: ABCB10A-c007 |
GI Number: 9961244 |
Vector: pFB-LIC-Bse. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ] |
DNA sequence: |
Expressed sequence (small letters refer to tag sequence): |
Tags and additions: N-terminal, TEV cleavable hexahistidine tag. (^ cleavage site) |
Host: Spodoptera frugiperda (SF9) insect cells |
Growth Medium & Induction Protocol: Insect cells with a density of 2x106 per litre of cell culture in SF900 medium (Invitrogen) were infected with recombinant baculovirus (5ml P2 virus per litre cell culture) and incubated for 72 hours. Cells were harvested by centrifugation and the pellet was flash frozen in liquid N2. |
Extraction buffer, extraction method: Frozen pellets were thawed and re-suspended in hypotonic buffer for lysis using a dounce homogeniser and DIAX homogeniser (Heidolph) at 10,000 r.p.m for 2 minutes. Cell membranes were collected by ultracentrifugation at 100,000Xg and the homogenisation repeated. The membranes were further washed with hypertonic buffer twice and the washed membranes were re-suspended in extraction buffer supplemented with 1% DDM, 0.1% CHS and incubated at 4°C with stirring. The extracted protein was separated from insoluble membranes by ultracentrifugation and collected for purification. |
Hypotonic buffer: 10mM HEPES pH7.5, 0.5 mM EDTA Hypertonic buffer: 10mM HEPES pH7.5,1M NaCl, 0.5mM EDTA Extraction/wash buffer: 50 mM HEPES, pH 7.5; 200 mM NaCl; 20 mM imidazole, 0.5mM MgCl2 and 0.5 mM TCEP |
Column 1: Co-affinity. Cobalt Talon (Clontech), 4 ml of 50 % slurry in 1.5 x 10 cm column, washed with extraction/ wash buffer. |
Column 1 Buffers: |
Column 1 Procedure: The solubilised membrane protein was batch bound to Cobalt Talon resin equilibrated with extraction buffer at 4°C for one hour and subsequently poured into a gravity flow glass column. The column was then washed with 25CV of wash buffer followed by elution in 1CV fractions until all the protein was eluted. |
Enzymatic Treatment: 1mg TEV protease was added overnight at 4°C to purified protein with concurrent buffer exchange by dialysis using a 3kDa cut off dialysis membrane into gel filtration buffer. The protease was removed and tag cleaved protein was collected by binding to Cobalt talon and collecting the flow-through. |
Column 2: Size Exclusion Chromatography. Superose6 (GE healthcare) |
Column 2 Buffer: |
Column 2 Procedure: The protein was concentrated and applied to a Superose6 gel filtration column equilibrated with gel filtration buffer using an AKTA Prime system. |
Mass spec characterization: |
Protein Concentration: Protein was concentrated to >8mg/ml using a 100kDa cut-off concentrator and back diluted to 5-8mg/ml with the addition of 2mM Mg-AMP-PNP. |
Crystallization: Crystals were grown in sitting drops at 20°C. Drops (200nl) comprising protein solution (5mg/ml; ABCB10A DDM/CHS/AMP-PNP) and reservoir solution (0.1-0.2 M NaCl, 5-7% (v/v) jeffamine M600, 30-40% (v/v) PEG400, 0.1 M glycine pH 9.5) in protein:reservoir ratios of 2:1 or 1.5:1 were equilibrated against 20µl of the same reservoir solution. Crystal plates were transferred to 6°C prior to directly flash cooling crystals in liquid nitrogen. |
Data Collection: |
Structure Solution: Initial phases were calculated using SIRAS. Two Hg sites were located with SHELXD and phase refinement was carried out with SHARP / SOLVE. Phase extension / density modification to 3.4Å yielded an interpretable map and allowed an initial backbone trace to be built. However, both data anisotropy and disorder of the NBD hindered model completion using the plate form crystals. The structure was finally solved using the refined, higher resolution rod-form A structure. Molecular replacement was used to position the separate transmembrane and nucleotide-binding domains. Refinement was carried out with autoBUSTER using LSSR restraints to the higher resolution rod-form A structure. |