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Materials and Methods: HSPA5 (3IUC)

StructureHSPA5
PDB Code3IUC
Entry clone accessionBC020235
Entry clone sourceMammalian Gene Collection
SGC clone accessionHSPA5A-k010
TagN-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*sm
Construct sequenceMHHHHHHSSGVDLGTENLYFQSMDVGTVVGIDLGTTYSCVGVFKNGRVEIIANDQGNRITPSYVAFTPEGERLIGDAAKNQLTSNPENTVFDAKRLIGRTWNDPSVQQDIKFLPFKVVEKKTKPYIQ
VDIGGGQTKTFAPEEISAMVLTKMKETAEAYLGKKVTHAVVTVPAYFNDAQRQATKDAGTIAGLNVMRIINEPTAAAIAYGLDKREGEKNILVFDLGGGTFDVSLLTIDNGVFEVVATNGDTHLGGE
DFDQRVMEHFIKLYKKKTGKDVRKDNRAVQKLRREVEKAKRALSSQHQARIEIESFYEGEDFSETLTRAKFEELNMDLFRSTMKPVQKVLEDSDLKKSDIDEIVLVGGSTRIPKIQQLVKEFFNGKE
PSRGINPDEAVAYGAAVQAGVLSGDQD
VectorpNIC-Bsa4
Expression hostBL21(DE3) R3 pRARE, where R3 denotes a derivative of BL21(DE3) resistant to a strain of T1 bacteriophage (SGC Oxford) and the pRARE plasmid originating from the Rosetta strain (Novagen) supplies tRNAs for rare codons.
Growth methodCells from a glycerol stock were grown in 20 mL TB supplemented with 8 g/l glycerol, 100 µg/mL kanamycin and 34 µg/mL chloramphenicol at 30 ºC overnight. The overnight culture (20 mL) was used to inoculate 1.5 l TB supplemented with 8 g/l glycerol, 50 µg/mL kanamycin and approximately 200 µl 204 Antifoam A6426 (Sigma). The culture was grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 ºC until OD600 reached ~2. The culture was down-tempered to 18 ºC over a period of 1 hour before target expression was induced by addition of 0.5 mM IPTG. Expression was allowed to continue overnight and cells were harvested the following morning by centrifugation (4,400 x g, 10 min, 4 ºC). The resulting cell pellet (43 g wet cell weight) was resuspended in lysis buffer (1.5 mL/g cell pellet), supplemented with 2000 U Benzonase (Merck) and one tablet of Complete EDTA-free protease inhibitor (Roche Applied Science). The cell suspension was stored at -80 ºC.
Extraction buffersLysis buffer: 100 mM HEPES, 500 mM NaCl, 10% glycerol, 10 mM imidazole, 0.5 mM TCEP, pH 8.0.
Extraction procedureThe cell suspension was quickly thawed in water. Cells were disrupted by sonication (Vibra-Cell, Sonics) at 80% amplitude for 3 min effective time (pulsed 4s on, 4s off) and cell debris was removed by centrifugation (49,000 x g, 20 min, 4 ºC). The supernatant was decanted and filtered through a 0.45 µm flask filter.
Purification buffersIMAC wash1 buffer: 20 mM HEPES, 500 mM NaCl, 10% glycerol, 10 mM imidazole, 0.5 mM TCEP, pH 7.5
IMAC wash2 buffer: 20 mM HEPES, 500 mM NaCl, 10% glycerol, 25 mM imidazole, 0.5 mM TCEP, pH 7.5
IMAC elution buffer: 20 mM HEPES, 500 mM NaCl, 10% glycerol, 500 mM imidazole, 0.5 mM TCEP, pH 7.5
Gel filtration (GF) buffer: 20 mM HEPES, 300 mM NaCl, 10% glycerol, 0.5 mM TCEP, pH 7.5
Purification procedureColumns
IMAC: Ni-charged 1 mL HiTrap Chelating HP (GE Healthcare)
Gel filtration column: HiLoad 16/60 Superdex 200 Prep Grade (GE Healthcare)

Procedure
MAC columns were equilibrated with IMAC wash1 buffer, and gel filtration columns were equilibrated with GF buffer. Purification of the protein was performed on an ÄKTAxpress system (GE Healthcare). The filtered lysate was loaded onto the Ni-charged HiTrap Chelating column and washed with IMAC wash1 buffer followed by IMAC wash2 buffer. Bound protein was eluted from the IMAC column with IMAC elution buffer and automatically loaded onto the gel filtration column. Fractions containing the target protein were identified by SDS-PAGE, pooled, and fresh TCEP was added to a final concentration of 2 mM. The protein was concentrated using an Amicon Ultra-15 centrifugal filter device (10,000 NMWL; Millipore) to 31 mg/mL in a volume of 0.3 mL. The identity of the protein was confirmed by mass spectrometry.

CrystallizationCrystals were obtained by the sitting drop vapour diffusion method in a 96-well plate. 0.1 µl protein solution (31 mg/mL) including 5 mM ADP and 5 mM MgCl2 was mixed with 0.1 µl of well solution consisting of 20% PEG6000, 0.2 M CaCl2, 0.1 M NaAcetate pH 5. The plate was incubated at 20 ºC. For data collection the crystals were quickly transferred to a cryo solution consisting of well solution complemented with 20% glycerol, and flash frozen in liquid nitrogen.
Data collectionDiffraction data to 2.13 Å resolution was collected at BESSY BL14-1, Berlin, Germany
Data processingThe structure was solved by molecular replacement using HSPA2 (PDB: 3I33) as template. The space group was P1 with cell dimensions a=48.21 Å, b=94.99 Å, c=51.79 Å, α=98.85°, β=94.88°, γ=117.61°. The asymmetric unit consisted of two polypeptide chain with two ADP, and Ca2+ ion located in the active site of the enzyme. The structure was refined with REFMAC5. Data in the interval 29.1-2.4 Å resolution was used and at the end of the refinement the R values were: R=19.4% and Rfree=27.0%. Coordinates for the crystal structure were deposited in the Protein Data Bank, accession code 3IUC.

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