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Materials and Methods: LMSAH (3G1U)

StructureLMSAH
PDB Code3G1U
Entry clone accessionQ4Q124
Entry clone sourcein-house cloning
SGC clone accessionLMSAHA-k006
TagN-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*sm
Construct sequencemhhhhhhssgvdlgtenlyfq*smADYKVKDISLAEWGRKAIELAENEMPGLMELRREYGPSQPLKGAKIAGCLHMTVQTAVLIETLKALGAELRWSSCNIFSTQDNAAAAIAKTGVPVFAWKGETD
EEYEWCIAQTVKGFSGDGLPNMILDDGGDLTNLVIDRYPELVPKIFGISEETTTGVKNLYKRLSKGNLPISAINVNDSVTKSKFDNLYGCRESLVDGIKRATDVMIAGKTCCVCGYGDVGKGCAAAL
RAFGARVVVTEVDPINALQASMEGYQVALVEDVMADAHIFVTTTGNDDIITSDHFPHMRDDAIVCNIGHFDTEIQVGWLEANAKEHVEIKPQVDRYTMENGRHIILLAKGRLVNLGCASGHPSFVMS
NSFTNQVLAQIELWSNRDNGKYPRGDKAGVFFLPKALDEKVAALHLAHVGAKLTKLTPKQAEYINCPVNGPFK
VectorpNIC-Bsa4
Expression hostE.coli BL21(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 0.75 mL 204 Antifoam A6426 (Sigma). The culture was grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 ºC until OD600 reached ~2. The bottle 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 (20 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 briefly 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 (49,100 x g, 20 min, 4 ºC). The supernatant was decanted and filtered through 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
Purification of the protein was performed as a two step process on an ÄKTAxpress system (GE Healthcare). Prior to purification, columns were equilibrated with IMAC wash1 buffer and gel filtration buffer, respectively. 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 pooled and fresh TCEP was added to a final concentration of 2 mM. The protein was subsequently concentrated using an Amicon centrifugal filter device with 10,000 NMWL (Millipore) to 25 mg/mL in a volume of 2.4 mL.

Tag removal
The N-terminal histidine tag was proteolytically removed by incubating LMSAHA with His-tagged TEV protease (van den Berg, S., J. Biotech 121, 291-298 (2006)) at a molar ratio of 160:1 at 20 ºC overnight. The proteolytic reaction went to completion, as judged by SDS-PAGE. Target protein was purified from tag and protease by passing the reaction mixture over a Ni-charged 1 mL HiTrap Chelating HP column (GE Healthcare) pre-equilibrated with IMAC wash1 buffer. The cleaved protein was concentrated using a centrifugal filter device to 35 mg/mL, resulting in a volume of 0.5 mL, and subsequently dialyzed against GF buffer containing 2 mM TCEP. 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 of the protein solution (diluted to 25 mg/mL with GF buffer) including 3 mM NAD was mixed with 0.1 µl of well solution consisting of 0.1 M Bicine, pH 9 and 20% PEG 6000. The plate was incubated at 4 ºC and crystals appeared within 1 day. The crystals were quickly transferred to cryo solution containing well solution, 0.2 M NaCl and 20% glycerol and flash frozen in liquid nitrogen.
Data collectionData sets were collected on a single crystal to 2.2 Å resolution at DIAMOND (I03). This data used for the final refinement belonged to P1 space group with cell parameters of a= 72.38 Å, b=82.47 Å, c= 83.88 Å α=87.02 β=71.41 γ=74.00
Data processingData was integrated with XDS, scaled with XSCALE and the structure was solved using MOLREP with PDB ID = 1LI4 as a search model. Four chains were found in the asymetric unit. The model was improved by successive rounds of manual model building in COOT and refinement with Refmac5. Final R-values were R= 18.15% and Rfree= 23.16%. Coordinates and structure factors were deposited in the PDB with accession code 3G1U.

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