Kronsteiner Barbara, Malissa Hans, Stutz Hanno
Division of Chemistry, Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
Electrophoresis. 2007 Jul;28(13):2241-51. doi: 10.1002/elps.200600764.
A preparation of recombinant birch pollen allergen of Betula verrucosa isoform 1a (Bet v 1a) containing chemically modified (carbamylated) variants has been analyzed by CZE and CIEF. In CZE, employing a 100 mmol/L MES buffer at pH 6.50, with 0.4 mmol/L tetraethylenepentamine (TEPA) added, allowed for the resolution of 17 protein fractions. The CIEF profiling of the allergen preparation required a combination of a wide-pH-range carrier ampholyte (CA) of pH 3-10 with two narrow-range CAs of pH 5-6 and 5-7. For CIEF, 91 mmol/L of glycine at pH 2.12 and 20 mmol/L of CHES at pH 10.00 were applied as anolyte and catholyte, respectively. The generated pH gradient was nonlinear with a flat slope for pH 4-6, thus providing an improved resolution. In CIEF, up to 18 protein fractions were distinguished as well. The pI of the target allergen Bet v 1a was 4.9 as determined by means of two pI marker compounds flanking the allergen. Relative purity of the target allergen within the preparation containing carbamylated variants was in accordance for both separation systems and varied between 40.7 and 42.8%.
对一种含有化学修饰（氨甲酰化）变体的重组疣皮桦花粉过敏原1a亚型（Bet v 1a）制剂进行了毛细管区带电泳（CZE）和毛细管等电聚焦（CIEF）分析。在CZE中，使用pH 6.50的100 mmol/L MES缓冲液，并添加0.4 mmol/L五乙烯六胺（TEPA），可分离出17个蛋白质组分。过敏原制剂的CIEF分析需要将pH 3 - 10的宽pH范围载体两性电解质（CA）与pH 5 - 6和5 - 7的两种窄范围CA结合使用。对于CIEF，分别将pH 2.12的91 mmol/L甘氨酸和pH 10.00的20 mmol/L 2-（环己氨基）乙磺酸（CHES）用作阳极电解液和阴极电解液。产生的pH梯度是非线性的，在pH 4 - 6范围内斜率平缓，从而提高了分辨率。在CIEF中，也区分出了多达18个蛋白质组分。通过位于过敏原两侧的两种pI标记化合物测定，目标过敏原Bet v 1a的pI为4.9。在含有氨甲酰化变体的制剂中，目标过敏原的相对纯度在两种分离系统中一致，在40.7%至42.8%之间。
