Mallu Maheswara Reddy, Vemula Sandeep, Ronda Srinivasa Reddy
Centre for Bioprocess Technology, Department of Biotechnology, KLEF University, Green Fields, Vaddeswaram, Guntur, Andhra Pradesh, 522 502, India.
3 Biotech. 2016 Jun;6(1):112. doi: 10.1007/s13205-016-0412-z. Epub 2016 May 17.
Antithrombin (AT) is a glycoprotein that inactivates the several physiological target enzymes of coagulation system. The effect of purification strategies plays a crucial role in getting maximum recovery of yield, purity and biological activity of recombinant human antithrombin (rhAT). In the present work, the task of purifying rhAT from Saccharomyces cerevisiae BY4741 has been carried out using two different approaches such as cross flow filtration (CFF) system and chromatography methods. In the first approach, the protein was concentrated and partially purified through CFF to achieve maximum recovery yield and purity of 87 and 94 %, respectively. In the second approach, purification involved a single step chromatography with various types of ion exchange and size exclusion resins to analyze the maximum rhAT recovery yield and purity. From the experimental results, it has been observed that the size exclusion chromatography (SEC) technique with Superose 12 matrix was suitable for the purification of rhAT and achieved the maximum recovery yield and purity of 51 and 97 %, respectively. Further, to acquire a high recovery yield and purity of rhAT, the effect of various chromatographic conditions such as mobile phase, mobile phase pH, flow rate, sample volume and sample concentration were also investigated. Under the optimal chromatographic conditions, rhAT was significantly recovered and purified in a single step with maximum recovery yield, purity and biological activity of 67, 99 % and 410 IU/L, respectively. Based on these investigations, it was concluded that SEC with Superose 12 matrix was a more suitable and a potential method for the purification of rhAT.
抗凝血酶（AT）是一种糖蛋白，可使凝血系统的几种生理靶酶失活。纯化策略的效果对于实现重组人抗凝血酶（rhAT）的最大产量回收率、纯度和生物活性起着至关重要的作用。在本研究中，已采用两种不同方法从酿酒酵母BY4741中纯化rhAT，即错流过滤（CFF）系统和色谱方法。在第一种方法中，通过CFF对蛋白质进行浓缩和部分纯化，以分别实现87%和94%的最大回收率和纯度。在第二种方法中，纯化涉及使用各种类型的离子交换和尺寸排阻树脂进行单步色谱分析，以分析rhAT的最大回收率和纯度。从实验结果可以看出，采用Superose 12基质的尺寸排阻色谱（SEC）技术适用于rhAT的纯化，分别实现了51%和97%的最大回收率和纯度。此外，为了获得高回收率和纯度的rhAT，还研究了各种色谱条件的影响，如流动相、流动相pH值、流速、样品体积和样品浓度。在最佳色谱条件下，rhAT在单步中显著回收和纯化，最大回收率、纯度和生物活性分别为67%、99%和410 IU/L。基于这些研究，得出结论：采用Superose 12基质的SEC是一种更合适且有潜力的rhAT纯化方法。
