RESUMO
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.
Assuntos
Saccharomyces cerevisiae/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/biossíntese , Antitrombina III/isolamento & purificação , Antitrombina III/biossíntese , Western Blotting , Cromatografia Líquida de Alta Pressão , Reatores Biológicos , Fermentação , FiltraçãoRESUMO
Among approaches applied to obtain high productivity and low production costs in bioprocesses are high cell density and the use of low cost substrates. Usually low cost substrates, as waste/agroindustrial residues, have low carbon concentration, which leads to a difficulty in operating bioprocesses. Real time control of process for intracellular products is also difficult. The present study proposes a strategy of repeated fed-batch with cell recycle to attain high cell density of Cupriavidus necator and high poly(3-hydroxybutyrate) (P(3HB)) productivity, using a substrate with low carbon source concentration (90 g l(-1)). Also, the use of the oxygen uptake rate data was pointed out as an on line solution for process control, once P(3HB) is an intracellular product. The results showed that total biomass (X), residual biomass (Xr) and P(3HB) values at the end of the culture were 61.6 g l(-1), 19.3 g l(-1) and 42.4 g l(-1) respectively, equivalent to 68.8 % of P(3HB) in the cells, and P(3HB) productivity of 1.0 g l(-1) h(-1). Therefore, the strategy proposed was efficient to achieve high productivity and high polymer content from a medium with low carbon source concentration.