Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800, Penang, Malaysia.
Protein J. 2010 Oct;29(7):509-15. doi: 10.1007/s10930-010-9281-1.
This paper describes a refinement in the purification step that facilitated the downstream recovery of high purity BmR1 recombinant protein, which is a protein used as a test reagent in the commercialized rapid tests for detection of lymphac filariasis i.e. Brugia Rapid™ and panLF rapid™. Purification was performed by immobilized metal affinity chromatography (IMAC), followed by ion exchange chromatography (IEX). Results showed that a total of 10.27 mg of BmR1 was obtained when IMAC was performed using 20 mM of imidazole and 5 column volume of wash buffer containing 500 mM of NaCl. Purity of the target protein was enhanced when buffer at pH 5.8 was used during the IEX. Two proteins that recurrently appeared below the BmR1 recombinant protein were identified by mass-spectrometry analysis as the same protein, thus they were probably degradation products of BmR1. These strategies improve purity of the target protein to be used in applications such as production of aptamers and monoclonal antibodies.
本文描述了一种改进的纯化步骤，该步骤有助于下游回收高纯度的 BmR1 重组蛋白，该蛋白用作商业化快速检测丝虫病的检测试剂，即 Brugia Rapid™ 和 panLF rapid™。纯化是通过固定化金属亲和层析（IMAC），然后是离子交换层析（IEX）进行的。结果表明，当 IMAC 使用 20mM 咪唑和 5 个柱体积的含有 500mM NaCl 的洗涤缓冲液进行时，可获得总共 10.27mg 的 BmR1。当在 IEX 期间使用 pH5.8 的缓冲液时，目标蛋白的纯度得到提高。通过质谱分析鉴定出在 BmR1 重组蛋白下方反复出现的两种蛋白为同一种蛋白，因此它们可能是 BmR1 的降解产物。这些策略可提高目标蛋白的纯度，用于生产适体和单克隆抗体等应用。
