Watkins Harriet A, Baker Edward N
Maurice Wilkins Centre for Molecular Biodiscovery and School of Biological Sciences, 3A Symonds Street, Private Bag 92019, Auckland, New Zealand.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Aug 1;64(Pt 8):746-9. doi: 10.1107/S1744309108021118. Epub 2008 Jul 31.
The predicted ribonuclease (RNase) HI domain of the open reading frame Rv2228c from Mycobacterium tuberculosis has been cloned as a hexahistidine fusion and a maltose-binding protein (MBP) fusion. Expression was only observed for the MBP-fusion protein, which was purified using amylose affinity chromatography and gel filtration. The RNase HI domain could be cleaved from the MBP-fusion protein by factor Xa digestion, but was very unstable. In contrast, the fusion protein was stable, could be obtained in high yield and gave crystals which diffracted to 2.25 A resolution. The crystals belong to space group P2(1) and have unit-cell parameters a = 73.63, b = 101.38, c = 76.09 A, beta = 109.0 degrees. Two fusion-protein molecules of 57,417 Da were present in each asymmetric unit.
结核分枝杆菌开放阅读框Rv2228c预测的核糖核酸酶（RNase）HI结构域已被克隆为六聚组氨酸融合体和麦芽糖结合蛋白（MBP）融合体。仅观察到MBP融合蛋白的表达，该蛋白通过直链淀粉亲和层析和凝胶过滤进行纯化。RNase HI结构域可通过因子Xa消化从MBP融合蛋白上切割下来，但非常不稳定。相比之下，融合蛋白稳定，能够高产获得，并得到了衍射分辨率达2.25 Å的晶体。这些晶体属于空间群P2(1)，晶胞参数为a = 73.63、b = 101.38、c = 76.09 Å，β = 109.0°。每个不对称单元中存在两个分子量为57,417 Da的融合蛋白分子。
