IMPROVED PURIFICATION METHOD OF RNA HELICASE OF HEPATITIS C VIRUS
PURPOSE: A purification method of RNA helicase from hepatitis C virus (HCV) is provided which results high yields and high purity of purified protease and saves times for the process of purification. CONSTITUTION: E. coli cells expressing Korean type HCV RNA helicase of which carboxy terminal having...
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| creator | LEE, HONG GYUN KANG, SEONG SU YUN, HYE SEONG JANG, HO JIN |
| description | PURPOSE: A purification method of RNA helicase from hepatitis C virus (HCV) is provided which results high yields and high purity of purified protease and saves times for the process of purification. CONSTITUTION: E. coli cells expressing Korean type HCV RNA helicase of which carboxy terminal having RNA helicase activity is isolated from NS 3 protein and tagged with histidine, are cultivated. The precipitated E.coli cells from centrifugation are suspended in the 20mM Tris buffer (pH 8.0-9.0) containing 0.1M NaCl and 1mM DTT or beta-mecapto ethanol and disintegrated by ultrasonication method. Supernatant of disintegrated cells is applied to Ni-NTA affinity column. Eluted protein with imidazole gradient is pooled and mixed with 20mM Tris buffer (pH 8.0) containing 0.1M NaCl and 1mM DTT. The mixture is applied to Q-Sepharose column and eluted with buffer having NaCl. The final yield of RNA helicase is 20mg/L and purity of protease is 99% based on capillary electrophoresis method. |
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CONSTITUTION: E. coli cells expressing Korean type HCV RNA helicase of which carboxy terminal having RNA helicase activity is isolated from NS 3 protein and tagged with histidine, are cultivated. The precipitated E.coli cells from centrifugation are suspended in the 20mM Tris buffer (pH 8.0-9.0) containing 0.1M NaCl and 1mM DTT or beta-mecapto ethanol and disintegrated by ultrasonication method. Supernatant of disintegrated cells is applied to Ni-NTA affinity column. Eluted protein with imidazole gradient is pooled and mixed with 20mM Tris buffer (pH 8.0) containing 0.1M NaCl and 1mM DTT. The mixture is applied to Q-Sepharose column and eluted with buffer having NaCl. 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CONSTITUTION: E. coli cells expressing Korean type HCV RNA helicase of which carboxy terminal having RNA helicase activity is isolated from NS 3 protein and tagged with histidine, are cultivated. The precipitated E.coli cells from centrifugation are suspended in the 20mM Tris buffer (pH 8.0-9.0) containing 0.1M NaCl and 1mM DTT or beta-mecapto ethanol and disintegrated by ultrasonication method. Supernatant of disintegrated cells is applied to Ni-NTA affinity column. Eluted protein with imidazole gradient is pooled and mixed with 20mM Tris buffer (pH 8.0) containing 0.1M NaCl and 1mM DTT. The mixture is applied to Q-Sepharose column and eluted with buffer having NaCl. 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CONSTITUTION: E. coli cells expressing Korean type HCV RNA helicase of which carboxy terminal having RNA helicase activity is isolated from NS 3 protein and tagged with histidine, are cultivated. The precipitated E.coli cells from centrifugation are suspended in the 20mM Tris buffer (pH 8.0-9.0) containing 0.1M NaCl and 1mM DTT or beta-mecapto ethanol and disintegrated by ultrasonication method. Supernatant of disintegrated cells is applied to Ni-NTA affinity column. Eluted protein with imidazole gradient is pooled and mixed with 20mM Tris buffer (pH 8.0) containing 0.1M NaCl and 1mM DTT. The mixture is applied to Q-Sepharose column and eluted with buffer having NaCl. The final yield of RNA helicase is 20mg/L and purity of protease is 99% based on capillary electrophoresis method.</abstract><edition>7</edition><oa>free_for_read</oa></addata></record> |
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| title | IMPROVED PURIFICATION METHOD OF RNA HELICASE OF HEPATITIS C VIRUS |
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