Cloning, expression, purification, crystallization and preliminary X-ray analysis of human liver glyceraldehyde-3-phosphate dehydrogenase.
Human liver glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified and crystallized using PEG 3350 as a precipitant. However, the crystals were extremely fragile towards osmotic shock. A 1% change in PEG 3350 content causes destruction of the crystals. After many trials for freezing the crystals, X-ray diffraction data from a native crystal were collected at 2.8 A resolution using as a cryoprotectant a mixture consisting of paraffin oil and Paratone-N in a 3:1 ratio and a cryoloop covered with Formver film. Crystals belong to space group P2(1), with unit-cell parameters a = 63.23, b = 97.84, c = 84.23 A, beta = 104.1 degrees. Molecular replacement with a starting model consisting of a homology model based on the low-resolution structure of human skeletal muscle GAPDH, which has 90% identical residues with the liver protein, led to a solution. Most of the current model was assigned properly in the electron-density map, but the map corresponding to some important regions containing the phosphate-binding loop was ambiguous. It is planned to crystallize human liver GAPDH in the presence of phosphate ions and/or some kind of inhibitor in order to fix the flexible region.
Warizaya
Masaichi
M
Exploratory Research Laboratories, Fujisawa Pharmaceutical Co. Ltd, 5-2-3 Tokodai, Tsukuba, Ibaraki 300-2698, Japan.
Kinoshita
Takayoshi
T
Kato
Akemi
A
Nakajima
Hidenori
H
Fujii
Takashi
T
D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008099:Liver / Q000201:enzymology*; D018482:Muscle, Skeletal / Q000201:enzymology; D011092:Polyethylene Glycols / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)