Dept. of Biological and Agricultural Engineering, Texas A&M University, 2117 TAMU, College Station, TX 77843, USA.
Biotechnol Prog. 2010 Sep-Oct;26(5):1303-11. doi: 10.1002/btpr.434.
Producing economically competitive recombinant human lysozyme from transgenic rice demands an inexpensive purification process for nonpharmaceutical applications. Human lysozyme is a basic protein, and thus, cation exchange chromatography was the selected method for lysozyme purification. Similar to other protein production systems, the identification of critical impurities in the rice extract was important for the development of an efficient purification process. Previous adsorption data indicated that phytic acid was probably responsible for an unacceptably low cation exchange adsorption capacity. In this study, we confirm that reducing phytic acid concentration improves lysozyme binding capacity and investigate alternative process conditions that reduce phytic acid interference. Compared with the previous best process, the adsorption capacity of human lysozyme was increased from 8.6 to 19.7 mg/mL when rice extract was treated with phytase to degrade phytic acid. Using tris buffer to adjust pH 4.5 extract to pH 6 before adsorption reduced phytic acid interference by minimizing phytic acid-lysozyme interactions, eliminated the need for phytase treatment, and increased the binding capacity to 25 mg/mL. Another method of reducing phytic acid concentration was to extract human lysozyme from rice flour at pH 10 with 50 mM NaCl in 50 mM sodium carbonate buffer. A similar binding capacity (25.5 mg/mL) was achieved from pH 10 extract that was clarified by acidic precipitation and adjusted to pH 6 for adsorption. Lysozyme purities ranged from 95 to 98% for all three processing methods. The tris-mediated purification was the most efficient of the alternatives considered.
从转基因水稻中生产具有经济竞争力的重组人溶菌酶需要一种用于非药物应用的廉价纯化工艺。人溶菌酶是一种碱性蛋白，因此，阳离子交换层析被选为溶菌酶纯化的方法。与其他蛋白质生产系统一样，鉴定水稻提取物中的关键杂质对于开发有效的纯化工艺非常重要。先前的吸附数据表明，植酸可能是导致阳离子交换吸附容量低的原因。在这项研究中，我们证实降低植酸浓度可以提高溶菌酶的结合能力，并研究了降低植酸干扰的替代工艺条件。与之前的最佳工艺相比，当用植酸酶处理水稻提取物以降解植酸时，人溶菌酶的吸附容量从 8.6 增加到 19.7mg/mL。在用 tris 缓冲液将 pH4.5 的提取物调节至 pH6 后进行吸附，可以通过最小化植酸-溶菌酶相互作用来减少植酸的干扰，无需植酸酶处理，并将结合能力提高至 25mg/mL。另一种降低植酸浓度的方法是在 pH10 下用 50mMNaCl 在 50mM 碳酸钠缓冲液中从水稻粉中提取人溶菌酶。通过酸性沉淀澄清 pH10 提取物，并将其调节至 pH6 进行吸附，可获得类似的结合能力（25.5mg/mL）。所有三种处理方法的溶菌酶纯度均在 95%至 98%之间。与所考虑的替代方法相比，tris 介导的纯化效率最高。
