Entry Clone Source: Origine

Entry clone accession: gi|18860900

SGC Construct ID: PTPRJA-c012

GenBank GI number: gi|18860900

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

Tags and additions: Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.

Final protein sequence:

The expressed sequence of 2CFV corresponds to the wild type sequence in which the WPD motif has been replaced by APD (highlighted in sequence in red). This mutant is still active using fluorescent based substrates:

mhhhhhhssgvdlgtenlyfq*smKLIRV
ENFEAYFKKQQADSNCGFAEEYEDLKLVG
ISQPKYAAELAENRGKNRYNNVLPYDISR
VKLSVQTHSTDDYINANYMPGYHSKKDFI
ATQGPLPNTLKDFWRMVWEKNVYAIIMLT
KCVEQGRTKCEEYWPSKQAQDYGDITVAM
TSEIVLPEWTIRDFTVKNIQTSESHPLRQ
FHFTSAPDHGVPDTTDLLINFRYLVRDYM
KQSPPESPILVHCSAGVGRTGTFIAIDRL
IYQIENENTVDVYGIVYDLRMHRPLMVQT
EDQYVFLNQCVLDIVRSQKDSKVDLIY

In addition we generated a mutant (2NZ6) in which the active site cysteine (marked in red) was substituted by a serine (C/S mutant):

mhhhhhhssgvdlgtenlyfq*smKLIRV
ENFEAYFKKQQADSNCGFAEEYEDLKLVG
ISQPKYAAELAENRGKNRYNNVLPYDISR
VKLSVQTHSTDDYINANYMPGYHSKKDFI
ATQGPLPNTLKDFWRMVWEKNVYAIIMLT
KCVEQGRTKCEEYWPSKQAQDYGDITVAM
TSEIVLPEWTIRDFTVKNIQTSESHPLRQ
FHFTSWPDHGVPDTTDLLINFRYLVRDYM
KQSPPESPILVHSSAGVGRTGTFIAIDRL
IYQIENENTVDVYGIVYDLRMHRPLMVQT
EDQYVFLNQCVLDIVRSQKDSKVDLIY

Host: BL21 (DE3)

Growth medium, induction protocol: 1ml from a 10 ml LB overnight culture containing 50 µg/ml kanamycin was used to inoculate 1 liter of LB media containing 50 µg/ml kanamycin. Cultures were grown at 37°C until the OD 600 reached ~0.3. After that the temperature was adjusted to 18°C. Expression was induced for 4 hours using 1mM IPTG at an OD 600 of 0.8. The cells were collected by centrifugation and the pellet resuspended in binding buffer and frozen. Binding buffer: 50mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazole, 5% glycerol.

Extraction buffer, extraction method: Cell pellets were lysed using a high pressure cell disrupter. The lysate was centrifuged at 50,000 rpm for 40 minutes and the supernatant collected for purification.

Column 1: Ni-affinity chromatography.

Buffers: Binding buffer: 50 mM HEPES pH 7.5, 500mM NaCl, 5% Glycerol. Wash buffer: 50 mM HEPES pH 7.5, 500mM NaCl, 20mM Imidazole, 5% glycerol. Elution buffer: 50mM HEPES pH 7.5, 500mM NaCl, 50 to 250mM Imidazole, 5% Glycerol.

Procedure: 5 ml of 50% Ni-NTA slurry (Qiagen) was applied to a 1.5 x 10 cm gravity column. The column was equilibrated with 30 ml binding buffer. The lysate was applied to the column which was subsequently washed with 3 x 10 ml wash buffer. PTPRJ was eluted by a step gradient generated by 5 ml portions of elution buffer with increasing concentration of imidazole (concentrations used: 50mM, 100mM, 250mM). The eluted protein fractions were collected and analyzed by SDS - PAGE . After elution DTT was added to a final concentration of 10mM.

Column 2: Size exclusion chromatography (Superdex S75, 60 x 1cm)

SEC -Buffers: 10 mM Hepes, pH 7.5, 25 mM NaCl.

Procedure: The fractions eluted of the Ni-affinity chromatography were pooled and concentrated to about 1 ml using Centricon concentrators (10kDa cut off). The concentrated protein was applied to a Superdex S75 column equilibrated in SEC buffer at a flow rate of 1 ml/min. Eluted fractions were 95% pure as judged by SDS - PAGE .

Protein concentration: Centricon with a 10kDa cut off in SEC -buffer

Crystallization:
W/A mutant (2CFV): Crystals were obtained using the vapor diffusion method and a protein concentration of 9 mg/ml by mixing 100nl of the concentrated protein with 100nl of a well solution containing 0.01M NiCl₂; 0.1M TRIS pH 8.5 and 1M Li₂SO₄. 1 mM of the peptide IGEHpYVHVNA corresponding to the Met-receptor were added to the protein prior to crystallization.

C/S mutant (2NZ6): Crystals were obtained using similar condition as described for the W/A mutant with a protein concentration of 9 mg/ml

Data Collection: Resolution: 2.5Å (2CFV) and 2.3Å (2NZ6); Crystals were cryo-protected using the well solution and 25% ethylene glycole and flash frozen in liquid nitrogen. X-ray source: Diffraction data were collected at the SLS beamline X10 at a single wavelength (0.979 Å).