Cp-GAPDH+NAD: Cryptosporidium parvum glyceraldehyde-3-phosphate dehydrogenase (cgd6_3790) in complex with NAD

Target ID:

cgd6_3790

Deposition Date:

Tuesday 1st April 2008

Families:

Oxidoreductases

Related Diseases:

Parasitic disease

Structure type:

protein-ligand

Download Coordinates:

3CPS

Authors:

Wernimont AK, Lew J, Kozieradzki I, Cossar D, Schapira M, Bochkarev A, Arrowsmith CH, Bountra C, Weigelt J, Edwards AM, Hui R, Pizarro J, Hills T

Site:

Toronto

3CPS

tabs

Structure Details

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH or G3PDH) is an enzyme involved in the ubiquitous process of glycolysis.
The parasite Cryptosporidium parvum appears to have the full complement of glycolytic enzymes including hexokinase (cgd6_3800),
glucose 6-phosphate isomerase (cgd2_3200), fructose biphosphate aldolase (cgd1_3020), trisephosphate isomerase (cgd1_3040) and GAPDH (cgd6_3790).

Specifically, GAPDH catalyzes the simultaneous phosphorylation and oxidation of glyceraldehyde 3-phosphate to 1-3 biphosphoglycerate, using NAD+ as the electron acceptor.

Materials and Methods

Cp-GAPDH

PDB:3CPS

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:cgd6_3790
Entry Clone Source:
SGC Clone Accession:cgd6_3790:T4-L339:D4
Tag:mhhhhhhssgrenlyfqg
Host:BL21 (DE3) Ros-Ox

Construct

Prelude:
Sequence:TLGINGFGRIGRLVLRACMERNDITVVAINDPFMDVEYMAYLLKYDSVHGNFNGTVEVSGKDLCINGKVVKVFQAKDPAEIPWGASGAQIVCESTGVFTTEEKASLHLKGGAKKVIISAPPKDNVPMYVMGVNNTEYDPSKFNVISNASCTTNCLAPLAKIINDKFGIVEGLMTTVHSLTANQLTVDGPSKGGKDWRAGRCAGNNIIPASTGAAKAVGKVIPALNGKLTGMAIRVPTPDVSVVDLTCKLAKPASIEEIYQAVKEASNGPMKGIMGYTSDDVVSTDFIGCKYSSIFDKNACIALNDSFVKLISWYDNESGYSNRLVDLAVYVASRGL
Vector:p28aLIC

Growth

Medium:TB
Antibiotics:
Procedure:The protein was expressed in the host cell in Terrific Broth (TB) in the presence of ampicillin/chloramphenicol (50 microgram/mL and 25 microgram/mL, respectively). A single colony was inoculated into 10 mL of LB with of ampicillin/chloramphenicol (50 microgram/mL and 25 microgram/mL respectively) in a 50 mL Falcon tube and incubated with shaking at 250 rpm overnight at 37 °C. The culture was transferred into 50 mL of TB with 50 microgram/mL kanamycin in a 250 mL shaking flask and incubated at 37 °C for 3 hours. Then the culture was transfer into 1.8 L of TB with 50 microgram/mL kanamycin and 0.3 mL of antifoam (Sigma) in a 2 L bottle and cultured using the LEX system to an OD600 > 5, cooled to 15 °C, and induced with 0.5 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15 °C.

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 1.0 - 2.5 mL 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. 5 mM DTT and 1 mM EDTA was added to the eluted CP-GAPDH (cgd6_3790) then it was loaded onto a superdex 200 gel filtration column. The eluted protein( in HEPES, pH 7.5 and 500 mM NaCl) was then concentrated using a 15 mL Amicon Ultra centrifugal filter device (Millipore). Stock concentration for CP-GAPDH (cgd6_3790) is 15 mg/mL .

Extraction

Procedure
The culture was harvested by centrifugation. Pellets from 4 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:28.9 mg/ml
Ligand
MassSpec:expected mass = 36732.6 Da, measured mass = 36733.4 Da
Crystallization:25% glycerolThe protein was crystallized at 20 degC in 20% Peg 3350, 0.2 M Tri-lithium citrate using the Sitting drop method.
NMR Spectroscopy:
Data Collection:
Data Processing: