Cherish Babu P V, Srinivas V K, Krishna Mohan V, Krishna Ella
Bharat Biotech International Limited, Genome Valley, Hyderabad 500078, India.
J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Jan 15;861(2):218-26. doi: 10.1016/j.jchromb.2007.10.008. Epub 2007 Oct 13.
Recombinant protein purification is facilitated using high expression systems which produce larger quantities of streptokinase protein as inclusion bodies. As the accumulation of active streptokinase is toxic to the host cells, we have optimized the conditions to achieve large amounts of streptokinase in the form of inclusion bodies. The solubility and yield of pure protein are highly dependent on various combinations of chemical additives, ionic and non-ionic detergents and salts, with solubilizing agents followed by refolding of denatured protein into active form. As the extraction of the purified streptokinase from inclusion bodies requires denaturation and a subsequent refolding step, careful balancing steps were needed to develop under different controlled conditions. Here the purified fragments of refolded proteins were screened to select the conditions that yield the active streptokinase having native conformation. The maximum specific activity of the purified streptokinase was achieved by these methods. The refolded recombinant streptokinase was analyzed by RP-HPLC showing a purity of 99%. Size exclusion chromatography profile shows that there are minimal aggregates in the active streptokinase protein and the percentage of renaturation is around 99%.
使用高表达系统有助于重组蛋白的纯化，该系统可产生大量作为包涵体的链激酶蛋白。由于活性链激酶的积累对宿主细胞有毒，我们优化了条件以获得大量包涵体形式的链激酶。纯蛋白的溶解度和产量高度依赖于化学添加剂、离子和非离子洗涤剂以及盐的各种组合，先使用增溶剂，然后将变性蛋白重折叠成活性形式。由于从包涵体中提取纯化的链激酶需要变性及随后的重折叠步骤，因此需要在不同的受控条件下仔细平衡步骤。在此，对重折叠蛋白的纯化片段进行筛选，以选择产生具有天然构象的活性链激酶的条件。通过这些方法实现了纯化链激酶的最大比活性。通过反相高效液相色谱法（RP-HPLC）分析重折叠的重组链激酶，纯度为99%。尺寸排阻色谱图显示活性链激酶蛋白中的聚集体最少，复性百分比约为99%。
