Expression and gene modification of Taq- polymerase using the SUMO purification system
Poster Number
73
Format
Poster Presentation
Faculty Mentor Name
Jerry Tsai
Faculty Mentor Department
Chemistry
Abstract/Artist Statement
Taq DNA polymerase is an enzyme produced by the thermophilic bacterium Thermus aquaticus, which is commonly used to amplify DNA in the polymerase chain reaction (PCR). The enzyme’s unique ability to withstand high temperatures, 36-75°C, makes it an efficient enzyme for cycles of heating and cooling. Purification of Taq polymerase can be done through heat lysis. While this is adequate for general use, this method produced an impure Taq polymerase mixed with other contaminating proteins. The impurities make it difficult to only study Taq polymers. With this in mind, an improved purification method was employed that combined the affinity tag of histidines along with the small ubiquitin-related modifier (SUMO) gene. This histidine tag of the SUMO system is an ideal purification technique for Taq polymerase due to the tag’s affinity to nickel. It was proposed that cloning the Taq gene into the SUMO expression system would facilitate easy purification. SUMO modulates protein structure and function by binding to the lysine side chains of target proteins. The Taq polymerase gene is cloned onto the C-terminal end of the SUMO gene using a PCR approach. Specific DNA primers were designed to introduce the excised Taq gene into the proper vector. Then, by inducing the bacterium, Escherichia coli (E. coli) with this gene, the Sumo-Taq protein can be expressed, to later be isolated using affinity chromatography involving the Nickel resin. Taq polymerase alone can be isolated by flowing SUMO protease through the column, resulting in simple and homogenous purification of this protein.
Location
Grave Covell
Start Date
21-4-2012 10:00 AM
End Date
21-4-2012 12:00 PM
Expression and gene modification of Taq- polymerase using the SUMO purification system
Grave Covell
Taq DNA polymerase is an enzyme produced by the thermophilic bacterium Thermus aquaticus, which is commonly used to amplify DNA in the polymerase chain reaction (PCR). The enzyme’s unique ability to withstand high temperatures, 36-75°C, makes it an efficient enzyme for cycles of heating and cooling. Purification of Taq polymerase can be done through heat lysis. While this is adequate for general use, this method produced an impure Taq polymerase mixed with other contaminating proteins. The impurities make it difficult to only study Taq polymers. With this in mind, an improved purification method was employed that combined the affinity tag of histidines along with the small ubiquitin-related modifier (SUMO) gene. This histidine tag of the SUMO system is an ideal purification technique for Taq polymerase due to the tag’s affinity to nickel. It was proposed that cloning the Taq gene into the SUMO expression system would facilitate easy purification. SUMO modulates protein structure and function by binding to the lysine side chains of target proteins. The Taq polymerase gene is cloned onto the C-terminal end of the SUMO gene using a PCR approach. Specific DNA primers were designed to introduce the excised Taq gene into the proper vector. Then, by inducing the bacterium, Escherichia coli (E. coli) with this gene, the Sumo-Taq protein can be expressed, to later be isolated using affinity chromatography involving the Nickel resin. Taq polymerase alone can be isolated by flowing SUMO protease through the column, resulting in simple and homogenous purification of this protein.