[Preparation of metal chelate affinity chromatographic medium and its application in the purification of 6 x histidine-tagged protein].
Using Sepharose CL-6B as support, 3-Chloro-1, 2-epoxypropane as activated agent, 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 6 x His-tagged fusion proteins. The amount of Co-CM-Asp-Sepharose reacted with 200 microL of lysate, the incubation time, wash condition and the imidazole concentration in the elution buffer were optimized. The purification results using Co-CM-Asp-Sepharose and Ni-NTA-Agarose (product of Qiagen) were compared. The CD155D1 fusion protein was also purified from 5mL of lysate and the amount of protein was determined by Bradford method. The results show that 60 microL of Co-CM-Asp-Sepharose (50% suspension) was suitable for the protein purification from 200 microL of lysate, the optimal incubation time of medium and lysate was 30 min, the optimal imidazole concentration in the eluting buffer was 200 mmol/L, and 200 microg of fusion protein was obtained. In a big scale experiment, 4.6 mg of fusion protein was obtained from 5 mL of lysate using 1.5 mL of Co-CM-Asp-Sepharose (50% suspension). Compared with Ni-NTA-Agarose, the Co-CM-Asp-Sepharose medium exhibits higher selectivity and the protein possesses higher purity.
Biochip Research and Development Center, Northwest University (National Engineering Research Center for Miniaturized Detection System), Xi' an 710069, China.
Shu-Juan
SJ
Li
Yong-Liang
YL
Sun
Dao-Dao
DD
Hu
Chao
C
Chen
Ya-Li
YL
Cui
D001224:Aspartic Acid / Q000737:chemistry*; D002614:Chelating Agents / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D004852:Epoxy Compounds / Q000737:chemistry; D006639:Histidine / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011108:Polymers / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D012685:Sepharose / Q000737:chemistry
D002614:Chelating Agents; D004852:Epoxy Compounds; D011108:Polymers; D011993:Recombinant Fusion Proteins; C036491:1-chloro-1,2-epoxypropane; D001224:Aspartic Acid; D006639:Histidine; D012685:Sepharose