Entry Clone Source: MGC

Entry Clone Accession: IMAGE:4824065

SGC Construct ID: PMP2A-c002

GenBank GI number: gi|4505909

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

Amplified construct sequence:
CATATGCACCATCATCATCATCATTCTTCT
GGTGTAGATCTGGGTACCGAGAACCTGTAC
TTCCAATCCATGAACAAATTCCTGGGCACC
TGGAAACTTGTCTCTAGTGAGAACTTTGAC
GATTACATGAAAGCTCTGGGTGTGGGGTTA
GCCACCAGAAAACTGGGAAATTTGGCCAAA
CCCACTGTGATCATCAGCAAGAAAGGAGAT
ATTATAACTATACGAACTGAAAGTACCTTT
AAAAATACAGAAATCTCCTTCAAGCTAGGC
CAGGAATTTGAAGAAACCACAGCTGACAAT
AGAAAGACCAAGAGCATCGTAACCCTGCAG
AGAGGATCACTGAATCAAGTGCAGAGATGG
GATGGCAAAGAGACAACCATAAAGAGAAAG
CTAGTGAATGGGAAAATGGTAGCGGAATGT
AAAATGAAGGGCGTGGTGTGCACCAGAATC
TATGAGAAGGTCTAACAGTAAAGGTGGATA
CGGATCCGAA

Final protein sequence (tag sequence in lowercase)
mhhhhhhssgvdlgtenlyfqsmNKFLGTW
KLVSSENFDDYMKALGVGLATRKLGNLAKP
TVIISKKGDIITIRTESTFKNTEISFKLGQ
EFEETTADNRKTKSIVTLQRGSLNQVQRWD
GKETTIKRKLVNGKMVAECKMKGVVCTRIY
EKV
mhhhhhhssgvdlgtenlyfqsm residues originate from the vector.

Tags and additions: N-terminal, TEV cleavable hexahistidine tag.

Host: E. coli BL21(DE3)-R3-pRARE2

Expression:10ul of BL21(DE3)-R3-pRARE2 glycerol stock were inoculated into 5ml of TB with 50ug/ml of kanamycin and 34ug/ml chloramphenicol and grown overnight at 37°C, 200rpm. 10ml of overnight culture were added to 1L of TB with 50ug/ml kanamycin and incubated at 37°C, 160rpm. After the OD₆₀₀ reached 1.0, the temperature was dropped to 18°C and 500ul of 1M IPTG was added to the final concentration of ~0.5mM. The culture was then incubated with shaking overnight at 18°C, 160rpm. The following morning the 4L culture was harvested and centrifuged for 10min at 4000rpm. Supernatant was discarded and cell pellets were resuspended in 80ml of a lysis buffer and frozen at -80°C.

Cell Extraction: Lysis buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5mM Imidazole, 5% glycerol + EDTA-free Complete (1 tablet/50ml).
The thawed cells were broken by 5 passes at 16.000 psi through a high pressure homogeniser followed by centrifugation for 45 min at 15.000rpm.

Purification:
Column 1: Ni-affinity, His-Trap, 1 ml (Amersham)
Column 2: Superdex 200, HiPrep 16/60 (Amersham)
Buffers: Start buffer: 50mM HEPES pH 7.5, 500mM NaCl, 20mM Imidazole, 5% glycerol, 1mM PMSF, 0.5mM TCEP; Washing buffer: 50mM HEPES pH 7.5, 500mM NaCl, 40mM Imidazole, 5% glycerol, 1mM PMSF, 0.5mM TCEP; Elution buffer: 50mM HEPES pH 7.5, 500mM NaCl, 5% glycerol, 250mM Imidazole, 0.5mM TCEP; GF buffer: 10mM HEPES pH 7.5, 500mM NaCl, 5% glycerol, 0.5mM TCEP
Procedure: The cell extract was loaded on the ÄKTA Express system. The extinction at 280nm was monitored and fractions were collected and analyzed by SDS-PAGE. Positive fractions were pooled and characterised by the mass spectrometry.

Concentration and buffer exchange: Using Amicon Ultra-15 concentrators with 10 kDa cutoff, the sample was concentrated to 11mg/ml. Concentrations were determined from the absorbance at 280 nm using NanoDrop.

Mass spectrometry characterization: Calculated mass of the construct was 17375 Da. The exact mass was confirmed by the mass spectrometry.

Crystallisation: Crystals were grown by vapor diffusion at 4°C in 150nl sitting drops. The drops were prepared by mixing 100nl of protein solution and 50nl of precipitant consisting of 3.15 M ammonium sulphate and 0.1 M sodium citrate pH 5.0. Ammonium sulphate acted as a cryo-protectant for flash-cooling of mounted crystal in liquid nitrogen.

Data collection: Resolution: 1.95 Å; X-ray source: Bruker in-house source