Production, purification and characterization of recombinant human antithrombin III by Saccharomyces cerevisiae

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.

Optimization of Fermentation Medium for the Production of Recombinant Human Antithrombin III from Saccharomyces cerevisiae through Statistical Experimental Designs.

This work, for the first time reports the optimization of recombinant human antithrombin (rhAT) production in Saccharomyces cerevisiae BY4741 using statistical experimental designs. Most applications of design of experiments (DoE) have concerned optimization of the composition of growth and production culture media. The typical objective is to identify a best selection and quantitative composition of significant medium supplements. In the present study, Plackett-Burman (PB) design followed by central composite design has been employed to evaluate and optimize the suitable culture medium for rhAT production. Influence of raffinose, glutamic acid and vitamin mixture were screened to be significant variables by PB design. The significant nutritional variables were further optimized using central composite design (CCD) for maximum production of rhAT. Central composite design (CCD) has been selected to explain the interaction effect of the three significant variables such as raffinose, glutamic acid, and vitamin mixture. The multiple regression equation (R2=0.9967) was used to optimize significant impact of medium component values to maximize rhAT formation. The optimized values were found to be 23.09765 g/L, 8.01816 g/L and 77.2056 mg/L for raffinose, glutamic acid and vitamin mixture, respectively. The maximum yield of rhAT of 38.97 μg/mL was obtained experimentally using CCD and was very close to the predicted response of 38.93 μg/mL.