Purifiction of Human Interleukin-5 (hIL-5)

• Main Author: 鍾宏彬
• Other Authors: 劉懷勝
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/21211151049252968694

碩士 === 國立臺灣大學 === 化學工程研究所 === 84 === Human interieukin-5 is a multifuntional cytokine which enhances the proliferation and differentiation of B cells and induces the eosinophil differentiation and the cytotoxic T-cell generation. Interieukin-5 is a kind of glycoprotein and its structure is a disulfide- linked homo-dimer with 115 amino acid residues in each chain. Bioassay told us that pure IL-5 has specific activity of 9.46x106 U/mg. The purification of recombinant human interieukin-5, produced in an insect cell/ baculovirus expression system, was investigated in our experiment. Serum free medium was used to conduct both the cell cultivation and the recombinant protein expression post virus infection. The highest IL-5 activity appeared at the 5th day after the virus infection. The culture broth was then used to proceed the following purification. In different temperature, pH, buffer and shear conditions, IL-5 showed crucial destruction due to the temperature increase and severely denatured under the pH higher than 8.6 or lower than 7.0. Having distant effect on IL-5 activity, 0.01% NaOH is the suitable sanitization/sterilization processing buffer. However, 30% ethylene glycol resulted in significant IL-5 denature in HIC process. Ultrafiltration caused the denature of IL-5 to a certain extent. The major reason for the loss of IL-5 activity is the shear stress from stirring. The higher speed the system used, the more IL-5 activity lost. It was found that the stirring rate of 90 rpm was the best to maintain most of the IL-5 activity. The selection of a series of suitable chrornatographic methods and the optimization for the purification process are main issues for discussion in purification engineering. Based on the SDS-PAGE assay, the products purified by HIC and AE still had other impurities. Finally, three steps including hydrophobic interaction chromatography, gel filtration chromatography, and anion exchange were employed to purify IL-5. Sample purified after the 3-step purification showed two clear bands of M.W. about 14 and 28 kDa. The specific activity of the final purified product was about 8.32x106units/mg. The activity loss in the 3-step process was about 50%. However, more than 30 fold of the purity increase was achieved. Affinity chromatography was also tested for IL-5 purification. Compared with the 3-step process, the recovery after affinity chromatography was only 39%, less than what was achieved in the 3-step process. But the specific activity obtained (8.9x106 units/ml) was a little higher. On the other hand, we replaced the AE operation with CE (cation exchange) in the 3-step process and also found two clear bands from SDS-PAGE analysis. However, the M.W. were about 15 and 30 kDa, higher than those from the 3-step process with AE. The higher M.W. is probably due to the inhomogeneity resulting from glycosylation. Finally, the scale up potential of the purification process was analyzed based on the purification results previously obtained. A batch of 50L culture broth was used as the basis for the purification consideration.