Fabry S, Lehmacher A, Bode W, Hensel R
Max-Planck-Institut für Biochemie, Martinsried, FRG.
FEBS Lett. 1988 Sep 12;237(1-2):213-7. doi: 10.1016/0014-5793(88)80204-7.
The gene of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the extremely thermophilic archaebacterium Methanothermus fervidus (growth optimum 82 degrees C) was cloned in vector pJF118EH and expressed in E. coli cells. As shown by molecular mass determination, protein sequencing, heat stability, and substrate saturation kinetics, the enzyme synthesized in E. coli is identical to the original enzyme from M. fervidus. The high thermostability of the E. coli-produced M. fervidus GAPDH allows rapid purification to homogeneity. From this enzyme protein crystals were grown which proved to be suitable for X-ray analysis. The crystals are of tetragonal space group P4(1)22 and contain a dimer per asymmetric unit.
从极端嗜热古细菌嗜热甲烷栖热菌（最适生长温度82摄氏度）中克隆了甘油醛-3-磷酸脱氢酶（GAPDH）基因，并将其克隆到载体pJF118EH中，在大肠杆菌细胞中表达。通过分子量测定、蛋白质测序、热稳定性和底物饱和动力学表明，在大肠杆菌中合成的酶与来自嗜热甲烷栖热菌的原始酶相同。大肠杆菌产生的嗜热甲烷栖热菌GAPDH的高热稳定性使得能够快速纯化至同质。从这种酶中生长出了适合X射线分析的蛋白质晶体。这些晶体属于四方晶系空间群P4(1)22，每个不对称单元包含一个二聚体。
