Power M E, Alm R A, Trust T J
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
J Bacteriol. 1992 Jun;174(12):3874-83. doi: 10.1128/jb.174.12.3874-3883.1992.
The flagellar filament-hook complex was removed from Campylobacter cells by shearing and was purified by differential solubilization and ultracentrifugation at pH 11 followed by cesium chloride buoyant density ultracentrifugation. Flagellar filaments were then dissociated in 0.2 M glycine-HCl (pH 2.2), and purified hooks were collected by ultracentrifugation. The hooks (105 by 24 nm) each displayed a conical protrusion at the proximal end, a concave cavity at the distal end, and helically arranged subunits. The apparent subunit molecular weight of the hook protein of seven of the eight Campylobacter strains studied was 92,500, while that of the other was 94,000. N-terminal amino acid analysis of the hook protein of two strains of Campylobacter coli and one strain of Campylobacter jejuni demonstrated that the first 15 residues were identical. Amino acid composition analysis showed that the Campylobacter hook protein contained 35.7% hydrophobic and 9.5% basic residues. Isoelectric focusing determined that the hook protein was acidic, with a pI of 4.9. Comparisons with the Salmonella and Caulobacter hook protein compositions and N-terminal amino acid sequences indicated that the Campylobacter protein was related, but more distantly than these two proteins were to each other. Immunochemical analysis with four different antisera and a panel of eight strains showed that serospecific epitopes were immunodominant. The Campylobacter hook proteins carried both cross-reactive and specific non-surface-exposed epitopes, as well as serospecific epitopes which were exposed on the surface of the assembled hook. One class of these surface-exposed hook epitopes was shared with serospecific flagellin epitopes and may involve posttranslational modification, while the second class of epitopes was hook specific and not shared with flagellin.
通过剪切从弯曲杆菌细胞中去除鞭毛丝-钩复合体，并通过在pH 11下进行差异溶解和超速离心，随后进行氯化铯浮力密度超速离心来纯化。然后将鞭毛丝在0.2 M甘氨酸-HCl（pH 2.2）中解离，并通过超速离心收集纯化的钩。这些钩（105×24 nm）在近端各显示一个锥形突起，在远端有一个凹腔，以及螺旋排列的亚基。所研究的8株弯曲杆菌中，有7株的钩蛋白表观亚基分子量为92,500，而另一株为94,000。对两株空肠弯曲杆菌和一株结肠弯曲杆菌的钩蛋白进行N端氨基酸分析表明，前15个残基是相同的。氨基酸组成分析表明，弯曲杆菌钩蛋白含有35.7%的疏水残基和9.5%的碱性残基。等电聚焦测定钩蛋白呈酸性，pI为4.9。与沙门氏菌和柄杆菌钩蛋白组成及N端氨基酸序列的比较表明，弯曲杆菌蛋白与之相关，但亲缘关系比这两种蛋白之间更远。用四种不同抗血清和一组八株菌株进行的免疫化学分析表明，血清特异性表位具有免疫显性。弯曲杆菌钩蛋白携带交叉反应性和特异性非表面暴露表位，以及组装钩表面暴露的血清特异性表位。这类表面暴露的钩表位中的一类与血清特异性鞭毛蛋白表位共享，可能涉及翻译后修饰，而第二类表位是钩特异性的，不与鞭毛蛋白共享。
