Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2078973 | Chinese Journal of Biotechnology | 2007 | 6 Pages |
Abstract
Using Sepharose CL-6B as support, 3-Chloro-1,2-epoxypropane as activated agent and carboxymethylated aspartate (CM-Asp) as chelating ligand, a chelate affinity chromatographic medium based on Co2+, named Co-CM-Asp-Sepharose, was prepared and used to purify 6ÃHis-tagged fusion proteins. The amount of Co-CM-Asp-Sepharose reacted with 200 μL of lysate, the incubation time, wash condition, and the imidazole concentration in the elution buffer were optimized. The purification results with Co-CM-Asp-Sepharose and commercialized Ni-NTA-Agarose were compared. The CD155D1 fusion protein was also purified from 5â¼mL of lysate and the amount of protein was determined by Bradford method. The results show that 60 μL of Co-CM-Asp-Sepharose (50% suspension) was suitable for protein purification from 200 μL of lysate, the optimal incubation time of medium and lysate was 30â¼min, the optimal imidazole concentration in the eluting buffer was 200â¼mmol/L, and 200 μg of fusion protein was obtained. In a large-scale experiment, 4.6â¼mg of fusion protein was obtained from 5â¼mL of lysate using 1.5â¼mL of Co-CM-Asp-Sepharose (50% suspension). Compared with Ni-NTA-Agarose, the Co-CM-Asp-Sepharose medium exhibits higher selectivity and the purified protein possesses higher purity.
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Authors
Shu-Juan LI, Yong-Liang SUN, Dao-Dao HU, Chao CHEN, Ya-Li CUI,