Efficient single step chromatographic purification of recombinant human antithrombin (rhAT) from Saccharomyces cerevisiae.
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.
Centre for Bioprocess Technology, Department of Biotechnology, KLEF University, Green Fields, Vaddeswaram, Guntur, Andhra Pradesh, 522 502, India.
Maheswara Reddy
MR
Mallu
Centre for Bioprocess Technology, Department of Biotechnology, KLEF University, Green Fields, Vaddeswaram, Guntur, Andhra Pradesh, 522 502, India.
Sandeep
S
Vemula
Centre for Bioprocess Technology, Department of Biotechnology, KLEF University, Green Fields, Vaddeswaram, Guntur, Andhra Pradesh, 522 502, India. rsr@kluniversity.in.
Srinivasa Reddy
SR
Ronda
Antithrombin; Chromatographic purification; Cross flow filtration; Ion exchange and size exclusion; Saccharomyces cerevisiae