Marchot P, Ravelli R B, Raves M L, Bourne Y, Vellom D C, Kanter J, Camp S, Sussman J L, Taylor P
Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636, USA.
Protein Sci. 1996 Apr;5(4):672-9. doi: 10.1002/pro.5560050411.
A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin-mAChE complex. Complexes of two distinct fasciculins, Fas1-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm x 0.5 mm x 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6(1)22 or P6(5)22 with unit cell dimensions a = b = 75.5 A, c = 556 A, giving a Vm value of 3.1 A3/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fas1-mAChE crystallizes in the same space group with identical cell dimensions.
通过在编码小鼠糖磷脂连接形式酶的cDNA的第549位插入一个早期终止密码子，产生了一种来自小鼠的可溶性单体形式的乙酰胆碱酯酶（mAChE），其羧基末端被截短。将该cDNA插入巨细胞病毒启动子后，在转染的HEK细胞中通过氨基糖苷抗性进行筛选，得到了能将大量mAChE分泌到培养基中的克隆。该酶以4.8 S的可溶性单体形式沉降。高水平的表达加上通过亲和层析进行的一步纯化，使我们能够对束状蛋白-mAChE复合物进行晶体学研究。在结晶之前形成了两种不同束状蛋白的复合物，即Fas1-mAChE和Fas2-mAChE，并对其进行了全面表征。尺寸达0.6 mm×0.5 mm×0.5 mm的单斜方晶体从pH 7.0范围内缓冲的硫酸铵溶液中自发生长。通过电泳迁移发现它们完全由复合物组成，并且衍射分辨率达到2.8 Å。对Fas2-mAChE晶体收集的初始X射线数据的分析确定空间群为P6(1)22或P6(5)22，晶胞尺寸a = b = 75.5 Å，c = 556 Å，Vm值为3.1 Å3/Da（或60%的溶剂），这与每个不对称单位中有一个Fas2-AChE复合物（72 kDa）分子一致。复合物Fas1-mAChE在相同的空间群中结晶，晶胞尺寸相同。
