Plasmodium berghei mitogen-activated protein kinase, PB000659.00.0

Target ID:

PP-MAPK2

Deposition Date:

Sunday 30th May 2010

Families:

Protein Kinase

Related Diseases:

Parasitic disease

Structure type:

apo

Download Coordinates:

3N9X

Authors:

Wernimont AK, Hutchinson A, Sullivan H, MacKenzie F, Kozieradzki I, Chau I, Lew J, Senisterra G, Artz J, Amani M, Cossar D, Bochkarev A, Arrowsmith CH, Edwards AM, Bountra C, Weigelt J, Hui R, Medicines for Malaria Ventur, Hills T

Site:

Toronto

ICM Datapack:

ICM Datapack

3N9X

tabs

Structure Details

The Plasmodium kinome features a small number of mitogen-activated kinases (MAPK), with MAP2 from Plasmodium falciparum having been found by means of northern blot analysis and immunofluorescence assays indicated to be expressed specifically in gametocytes responsible for transmission of the parasite to the mosquito vector (Dorin et al., 1999). Plasmodium MAP2 does not possess the conserved TXY activation motif usually found in MAPKs but instead features TSH in the same position. MAP2 from Plasmodium berghei is 82% identical to the P. falciparum orthologue. The purified sample of the full length protein was found to be dimeric in solution, but the construct leading to this structure was monomeric.

Materials and Methods

MAPK

PDB:3N9X

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:PB000659.00.0
Entry Clone Source:
SGC Clone Accession:PBG-PF11_0147:I110-S523:C2
Tag:mhhhhhhssgrenlyfqg
Host:BL21(DE3)-V2R-pACYC LamP

Construct

Prelude:
Sequence:IKNVHVPDNYIIKHLIGRGSYGYVYLAYDKNTEKNVAIKKVNRMFEDLIDCKRILREITILNRLKSDYIIRLYDLIIPDDLLKFDELYIVLEIADSDLKKLFKTPIFLTEEHIKTILYNLLLGENFIHESGIIHRDLKPANCLLNQDCSVKVCDFGLARTINSEKDTNIVNDLEENEEPGPHNKNLKKQLTSHVVTRWYRAPELILLQENYTKSIDIWSTGCIFAELLNMLQSHINDPTNRFPLFPGSSCFPLSPDRNSKKVHEKSNRDQLNIIFNIIGTPTEDDLKNINKPEVIKYIKLFPHRKPINLKQKYPSISDDGINLLESMLKFNPNKRITIDQALDHPYLKDVRKKKLENFSTKKIILPFDDWMVLSETQLRYIFLKEVQSFHPELVIPSVFTIHENNFYNNEKPSS
Vector:p15-mhl

Growth

Medium:TB
Antibiotics:
Procedure:Plasmodium berghei PBG-PF11_0147 was expressed in E. coli BL21(λDE3) V2R pACYC LamP in TB growth media in the presence of carbenicillin/chloramphenicol (100 microgram/mL and 34 microgram/mL, respectively). A single colony was inoculated into 25 mL of LB with of carbenicillin/chloramphenicol (100 microgram/mL and 34microgram/mL respectively) in a 50 mL Falcon tube and incubated with shaking at 250 rpm overnight at 37degC. Then the culture was transferred into 900 mls of TB with 100 microgram/mL Carbenicillin and 34 microgram/ml chloramphenicol , 0.3 mL of antifoam (Sigma), 9 mls of 0.83 M MgSO4 and trace elements in a 1L bottle and cultured using the LEX system to an OD600 of 5, cooled to 15°C, and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15degC.

Purification

Procedure
The cleared lysate was loaded onto a column prepacked with 10 g DE52 (Whatman) anion exchange resin (previously activated with 2.5 M NaCl and equilibrated with Binding Buffer); and subsequently onto a 2mL Ni-NTA (Qiagen) column pre-equilibrated with Binding Buffer at approximately 1 - 1.5 mL/min. The volume of the Ni-NTA resin was pre-determined by the predicted protein yield from test expression analysis. After the lysate was loaded, the DE52 was further washed with 20 mL of Binding Buffer. The Ni-NTA column was then washed with 200 mL of Wash Buffer at 2 - 2.5 mL/min. After washing, the protein was eluted with 15 mL of Elution Buffer. 1 mM TCEP and 1 mM EDTA was added to the eluted PBG-PF11_0147.

The protein sample was then loaded onto a superdex 200 gel filtration column. The eluted protein ( in 10 mM Hepes, pH 7.5 and 500 mM NaCl) was concentrated using a 15 ml Amicon Ultra centrifugal filter device (Millipore) with a 10 kDa cutoff. The protein was concentrated to 10 mg/ml using a 15 ml Amicon Ultra centrifugal filter device (Millipore) with a 10 kDa cutoff, then stored at 4 º C.

Extraction

Procedure
The culture was harvested by centrifugation. Pellets from 2 L of culture were resuspended to approximately 40 mL/L of cell culture in Binding Buffer with the addition of protease inhibitors (1 mM benzamidine and 1 mM phenylmethyl sulfonyl fluoride (PMSF)). Resuspended pellets stored at -80 degC were thawed overnight at 4 degC on the day before purification. Prior to mechanical lysis, each pellet from 1 L of culture was pretreated with 0.5 % CHAPS and 500 units of benzonase for 40 minutes at room temperature. Cells were mechanically lysed with a microfluidizer (Microfluidizer Processor, M-110EH) at approximately 18000 psi; and the cell lysate was centrifuged using a Beckman JA-25.50 rotor at ~75000 x g (24000 rpms) for 20 minutes at 10 degC.
Concentration:
Ligand
2 mM ATP, 4 mM MgCl2, 2 mM TCEPMassSpec:
Crystallization:Crystallization plate: IC-MAZ3MB:C8-1
buffer: 20% PEG 3350, 0.2 M tri-lithium citrate at 20 degC
Cryo conditions: crystallization condition +25% glycerol
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Dorin, D., Alano, P., Boccaccio, I., Ciceron, L., Doerig, C., Sulpice, R., and Parzy, D. (1999). An atypical mitogen-activated protein kinase (MAPK) homologue expressed in gametocytes of the human malaria parasite Plasmodium falciparum. Identification of a MAPK signature. J Biol Chem 274, 29912-29920.