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Entry Clone Source: Synthetic

Entry Clone Accession: n/a

SGC Construct ID: SEPX1A-c008

GenBank GI number: gi|7706511

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Amplified construct sequence:
CATATGCACCATCATCATCATCATTCTTCT
GGTGTAGATCTGGGTACCGAGAACCTGTAC
TTCCAATCCATGGAGGTTTTCCAGAATCAC
TTTGAACCTGGCGTTTACGTGTGTGCCAAG
TGTGGCTATGAGCTGTTCTCCAGCCGCTCG
AAGTATGCACACTCGTCTCCATGGCCGGCG
TTCACCGAGACCATTCACGCCGACAGCGTG
GCCAAGCGTCCGGAGCACAATAGATCTGAA
GCCTTGAAGGTGTCCTGTGGCAAGTGTGGC
AATGGGTTGGGCCACGAGTTCCTGAACGAC
GGCCCCAAGCCGGGGCAGTCCCGATTCTCA
ATATTCAGCAGCTCGCTGAAGTTTGTCCCT
AAAGGCAAAGAAACTTCTGCCTGACAGTAA
AGGTGGATACGGATCCGAA

Tags and additions: N-terminal Histidine-tag with TEV protease cleavage site

Final protein sequence (tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfqsMEVFQNHF
EPGVYVCAKCGYELFSSRSKYAHSSPWPAF
TETIHADSVAKRPEHNRSEALKVSCGKCGN
GLGHEFLNDGPKPGQSRFSIFSSSLKFVPK
GKETSA

Growth medium, induction protocol: 10ul of a glycerol stock was inoculated into 5ml of TB medium (supplemented with 50µg/ml Kanamycin, 34µg/ml Chloramphenicol) and cultured at 37°C o/n in a shaking incubator (250 rpm). Next day 0.75 ml of o/n culture was used to inoculate 1 litre of TB medium (6 x) and grown at 37°C with vigorous shaking (160 rpm) until the culture reaches an OD₆₀₀ of 1.6. Temperature was reduced to 18°C, and cells were induced with IPTG at a concentration of 0.3 mM, and further cultivated for 16 hrs. Cells were harvested by centrifugation at 6500 rpm for 10 min, and the cell pellet was stored at -20°C until further use.

Extraction buffer, extraction method:
Lysis buffer: 500 mM NaCl, 5% Glycerol, 50 mM HEPES pH 7.5, 5 mM Imidazole.
Complete® protease inhibitors (Roche, 1 tbl/50 ml).
Frozen cell pellets were thawed and resuspended in a total volume of 30-40 ml of lysis buffer, and disrupted by using sonicator, and a supernatant containing the target protein was obtained by centrifugation at 21,000 (rpm) for 45 minutes.

Column 1: Ni-Sepharose 6 Fast Flow

Column 1 Buffers:
Lysis buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 5 mM Imidazole.
Wash buffer: 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5% Glycerol, 30 mM Imidazole.
Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole
Note: All the buffers contain 0.5mM TCEP.

Column 2: SuperDex 75 16/60 HiLoad (GE/Amersham)

Column 2 Buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5 % glycerol, 0.5 mM TCEP.

Column 2 Procedure: The eluted protein from the Ni-affinity column was loaded on the gel filtration column in GF buffer at 1.0 ml/min on an AKTA Purifier system. Eluted proteins were collected in 1 ml fractions.

Enzymatic treatment: TEV cleaved.

Column 3: Ni-Sepharose (TEV clean up)

Column 3 Buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP

Column 3 Procedure: Total 25 mgs of protein was cleaved with 900 ug of TEV protease at 4 degree for 48 hours.

TEV clean up: The TEV cleaved protein was applied to a 1 ml Ni-Sepharose column, already equilibrated with gel filtration buffer (10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP). The flow through form the column was collected. The eluate from the column was monitored by SDS gel analysis.

Protein concentration: The target protein was concentrated to 10.2 mg/ml using Vivaspin 3K concentrators and stored at -80°C.