Hekmat Dariusch, Breitschwerdt Peter, Weuster-Botz Dirk
Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748, Garching, Germany,
Biotechnol Lett. 2015 Sep;37(9):1791-801. doi: 10.1007/s10529-015-1866-5. Epub 2015 May 26.
To investigate quantitatively and reproducibly a scalable, preparative crystallization method in novel stirred tanks using three different protein solutions containing residual microbial host cell proteins (HCP).
Lysozyme from solutions being spiked with up to 15% host cell proteins (HCP) (corresponding to 176,500 ppm) was crystallized within a 2.4-4.6 h at 93.7% yield using NaCl and glycerol. Lipase was crystallized under comparable conditions using NaCl and a mixture of two polyethylene glycols (PEG). Enhanced green fluorescent protein (eGFP) was overexpressed in E. coli yielding a solution containing 23% target protein. Residual HCP content after pre-treatment was 7-16%. eGFP was crystallized from these solutions within 1.75-4 h at 88.7% step yield using ethanol and the same mixture of two PEG as in the case of lipase. HCP contained in the solvent channels of the protein crystals could be removed by diffusive washing yielding final purities at or above 99%.
Preparative crystallization can be carried out with fast kinetics and high yields from solutions containing residual impurities and may represent an attractive alternative purification method compared to preparative chromatography, especially at large production scales.
使用三种含有残留微生物宿主细胞蛋白（HCP）的不同蛋白质溶液，在新型搅拌罐中对一种可扩展的制备性结晶方法进行定量且可重复的研究。
在添加了高达15%宿主细胞蛋白（HCP）（相当于176,500 ppm）的溶液中的溶菌酶，使用氯化钠和甘油在2.4 - 4.6小时内结晶，产率为93.7%。脂肪酶在类似条件下使用氯化钠和两种聚乙二醇（PEG）的混合物进行结晶。增强型绿色荧光蛋白（eGFP）在大肠杆菌中过表达，得到一种含有23%目标蛋白的溶液。预处理后的残留HCP含量为7 - 16%。使用乙醇以及与脂肪酶相同的两种PEG混合物，eGFP在1.75 - 4小时内从这些溶液中结晶，分步产率为88.7%。蛋白质晶体溶剂通道中含有的HCP可通过扩散洗涤去除，最终纯度达到或高于99%。
制备性结晶可以从含有残留杂质的溶液中以快速动力学和高收率进行，与制备性色谱相比，可能是一种有吸引力的替代纯化方法，尤其是在大规模生产中。
