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.

In vitro Propagation and GC-MS Studies of Ocimum basilicum Linn. var. pilosum (Willd.) Benth.

An in vitro propagation method is outlined for Ocimum basilicum Linn. var. pilosum (Willd.) Benth., a wild aromatic plant belongs to Lamiaceae family. Shoot buds were used as source of explants on MS media supplemented with different concentrations of growth regulators for callus growth, induction of multiple shoots and roots respectively. MS media with 1.5 mg/L of kinetin and 0.5 mg/L of NAA showed 95.5% shooting, maximum number of shoots (7.33) and relatively better shoot lengths (4.15 cm). Excised shoots were carefully transferred to halfstrength MS medium supplemented with 1.0 mg/L indole-3-butyric acid (IBA) for root induction and it yields 86.6% rooting. Whereas, average root length and number of roots observed were 1.73 cm and 3.31 respectively per explants. Rooted plantlets were hardened and successfully established in natural soil, where they grew and matured normally. GC-MS studies in methanolic leaf extract of naturally grown Ocimum species yielded 15 compounds. Two compounds viz. cis-9-Hexadecenal (35.06%) and n-Hexa decanoic acid (21.6%) accounted for the major share (56.66%). On the other hand, 2-hydroxy-6-methylbenzaldehyde (10.99%) as well as 4H-1-Benzopyran-4-one and 5-Hydroxy-6, 7-Dimethoxy-2-(4- Methoxyphenyl) (7.75%) represented only 18.74%. Establishment of reliable in vitro propagation protocol, with phytochemical profile of hitherto unreported Ocimum species further widens the scope to evaluate its therapeutic properties.

Aeration effect on Spirulina platensis growth and ã-linolenic acid production

The influence of aeration on algal growth and gamma-linolenic acid (GLA) production in a bubble column photobioreactor was investigated. Studies were performed in a 20-L reactor at different aeration rates (0.2-2.5 vvm). Static, continuous, and periodic operation of air resulted in 41.9%, 88.4%, and 108% air saturation of dissolved oxygen, for which the corresponding values of GLA were 2.3, 6.5, and 7.5 mg·g-1 dry cell weight, respectively. An increase in the aeration rate from 0.2 to 2.5 vvm enhanced both the specific growth rate and GLA content under periodic sparging in the bicarbonate medium. With a 6-fold increase in the aeration rate, the GLA content of the alga increased by 69.64% (5.6-9.5 mg· g-1 dry cell weight). In addition, the total fatty acid (TFA) content in dry biomass increased from 2.22% to 4.41%, whereas the algae maintained a constant GLA to TFA ratio within the aeration rate tested. The dependence of GLA production on the aeration rate was explained by interrelating the GLA production rate with the specific growth rate using the Luedeking and Piret mixed growth model.

Culture conditions stimulating high gama-Linolenic acid accumulation by Spirulina platensis / Condições de cultura simulando o levado acúmulo de ácido gama-linolênico por Spirulina platensis

Gamma-linolenic acid (GLA) production by Spirulina platensis under different stress-inducing conditions was studied. Submerged culture studies showed that low temperature (25ºC), strong light intensity (6 klux) and primrose oil supplement (0.8 percentw/v) induced 13.2 mg/g, 14.6 mg/g and 13.5 mg linolenic acid per gram dry cell weight respectively. A careful observation of fatty acid profile of the cyanobacteria shows that, oleic acid and linoleic acid, in experiments with varying growth temperature and oil supplements respectively, helped in accumulating excess γ-linolenic acid. In addition, cultures grown at increasing light regimes maintained the γ-linolenic acid to the total fatty acid ratio(GLA/TFA) constant, despite any change in γ-linolenic acid content of the cyanobacteria.

Estudou-se a produção de ácido γ-linolênico por Spirulina platensis em diferentes condições de estresse. Culturas submersas indicaram que temperatura baixa (25ºC), forte intensidade de luz (6 klux) e suplementação com óleo de prímula (0,8 por cento p/v) induziram a produção de ácido linolênico de 13,2 mg/g, 14,6 mg/g e 13,5 mg/g peso seco, respectivamente. Uma observação cuidadosa do perfil de ácidos graxos da cianobacteria indica que os ácidos oléico e linoléico, em experimentos com diferentes temperaturas de crescimento e suplementos de óleo, auxiliaram no acúmulo de excesso de ácido γ-linolênico. Além disso, as culturas obtidas em intensidades crescentes de luz mantiveram a relação ácido γ-linolênico/ácidos graxos totais constante, independentemente de qualquer mudança no conteúdo de ácido γ-linolênico da cianobactéria.