Jackson D G, Owen M J, Voorheis H P
Biochem J. 1985 Aug 15;230(1):195-202. doi: 10.1042/bj2300195.
A simple new technique was developed for the rapid purification of either the membrane-bound or the released forms of the variant surface glycoprotein of Trypanosoma brucei in high yield. Whole cells were used as the source of the membrane-bound form, and the supernatant of benzyl alcohol-treated cells was used as the source of the released form. The technique was based on extraction of the acid-treated protein into chloroform/methanol, followed by selective re-partition into aqueous salt solution. The yield of purified protein was found to be dependent critically on a low pH during the extraction/re-partition stages. This finding and the ability to cycle the protein repeatedly through organic and aqueous phases in a strictly pH-dependent manner suggested that the protein could undergo fully reversible denaturation/renaturation only while in an extensively protonated form. The yield was independent of the polarity of the organic phase and the protein concentration over a wide range. After purification, both forms retain their ability to react with specific antibody raised against the authentic native protein purified by conventional means. The amino acid composition and the identity of the N-terminal amino acid was the same for both forms of the protein. In addition, both forms had blocked C-terminal residues. There were determined to be 1.13 X 10(7) copies of the variant surface glycoprotein per cell.
开发了一种简单的新技术，可高产率快速纯化布氏锥虫变异表面糖蛋白的膜结合形式或释放形式。全细胞用作膜结合形式的来源，苄醇处理细胞的上清液用作释放形式的来源。该技术基于将酸处理的蛋白质提取到氯仿/甲醇中，然后选择性地重新分配到盐水溶液中。发现纯化蛋白的产量在提取/重新分配阶段关键取决于低pH值。这一发现以及蛋白质能够以严格的pH依赖性方式反复在有机相和水相之间循环的能力表明，该蛋白质只有在广泛质子化的形式下才能经历完全可逆的变性/复性。产量在很大范围内与有机相的极性和蛋白质浓度无关。纯化后，两种形式都保留了与针对通过传统方法纯化的真实天然蛋白产生的特异性抗体反应的能力。两种形式的蛋白质的氨基酸组成和N端氨基酸相同。此外，两种形式都有封闭的C端残基。确定每个细胞有1.13×10⁷个变异表面糖蛋白拷贝。
