Study On Preparation And Purification Of Protein

The key technology of proteomics is protein purification. Transferrin(Tf) is one of the trace protein, the concentration of which is 2.5 g/L in human plasma. Tf has many important functions such as antibacterial function, transporting iron and it could also be the growth factor of cell multiplication. More important, Tf contributes great research value in the field of cancer treatment. The current thesis takes Tf as target protein, created a two-step chromatography method of purifying Tf from Cohn fraction IV, which was discarded in the cold ethanol precipitation process. Tf production experiences the characterization and activity evaluation and Tf scale-up experiments were under optimum conditions.In the first place, the components, operation and working process of AKTA explorer 100 protein purification system were introduced, which laid the foundation for the subsequent protein purification process.Secondly, two-step chromatography method of purifying Tf from Cohn fraction IV was researched. It was found that Tris-HCl buffer is not good for the centrifugal of Cohn fraction IV in pretreatment process, which influenced the later filtration. Thus, we decided to dissolve Cohn fraction IV in ultra pure water. According to the differences in isoelectric point and hydrophobicity, we choose the purification scheme of the combination of ion exchange chromatography and hydrophobic chromatography. After comparing two important factors, the chromatographic media and pH in ion exchange chromatography process, it was discovered that Capto DEAE and pH7.0 were suitable for separation because they make the best separation and obtained Tf with good purity, high yield and purification speed. During the process of hydrophobic chromatography, salt concentration was chosen to be researched. Four different salt concentration effects were compared and it was discovered that 1.0 mol/L ammonium sulfate concentration contributes to good yield and purity.Next, the purified Tf experienced the characterization and activity evaluation. The final product was tested by HPSEC to estimate the integrity and the presence of aggregates. The results of circular dichroism(CD) showed that secondary structure of Tf has not been changed. The molecular weight of Tf which detected by mass spectra was 78726. The result of full wavelength scan showed that obvious characteristic absorption peak is found at 465 nm and that Tf can combine two irons. The antibacterial activity test displayed that Tf has obvious inhibition effect upon Escherichia coli.Finally, the scale-up experiment of Tf was conducted under the best chromatography conditions and more than 1 g Tf sample was obtained each batch. Tf purity reaches more than 99% and the yield increases to more than 80% during experiment.This thesis established a new two-step chromatography purification method, which has been proved to have high yield and purity, and this method is suitable for scale-up, saving valuable plasma resources by recycling wastes and providing convenience for the functional study of Tf.