Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae |
Mallu, Maheswara Reddy; Vemula, Sandeep & Ronda, Srinivasa Reddy
Background: Antithrombin III (ATIII) is a protein that inhibits abnormal blood clots (or coagulation) by breaking
down thrombin and factor Xa. ATIII helps to keep a healthy balance between hemorrhage and coagulation.
The present work demonstrated the production, purification and characterization of recombinant human
antithrombin (rhAT) from yeast Saccharomyces cerevisiae BY4741 was demonstrated. After expression of rhAT
by S. cerevisiae, the biomass and rhAT concentration were analyzed through fed-batch fermentation process.
Results: In fed-batch fermentation, the biomass (maximum cell dry weight of 11.2 g/L) and rhAT concentration
(312 mg/L) of the expressed rhAT were achieved at 84 h of cultivation time. The maximum cell lysis efficiency
(99.89%) was found at 8 s sonication pulse and 7 mL lysis buffer volume. The rhAT protein solution was
concentrated and partially purified using cross-flow filtration with the recovery yield and purity of 95
and 94%, respectively. The concentrated solution was further purified by the single step ion exchange
chromatography with the recovery yield and purity of 55 and ≥98%, respectively. The purified rhAT was
characterized by various analytical techniques, such as RP-HPLC, FT-IR, CD, SDS-PAGE, western blotting, and
Liquid chromatography mass spectrometry (LC–MS) analysis. The biological activity of rhAT was analyzed as
heparin cofactor to meet the therapeutic grade applications.
Conclusions: The simple, cost-effective and economically viable nature of the process used in the present study for
the production of rhAT will be highly beneficial for the healthcare sector. This may also be used to produce other
value-added therapeutic recombinant proteins expressed in S. cerevisiae, with greater effectiveness and ease.
Biological activity; Cell lysis; Cross flow filtration; Fed-batch fermentation; Purification; Secondary structure