Title

Analysis of Structure-Function Relationship in Taq DNA polymerase

Poster Number

46

Format

Poster Presentation

Abstract/Artist Statement

Taq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus. It is widely used in polymerase chain reaction (PCR). T. aquaticus was isolated from hot springs and Taq polymerase was identified as an enzyme able to withstand the thermal cycling conditions required for rapid amplification of DNA. Taq's optimum temperature for activity is 75-80°C, with a half-life of 9 minutes at 97.5°C.In current research, we used Taq polymerase to study the relationship between its structure and function based on the hypothesis that primary structure (amino acid sequence) codes for it’s folded 3D structure and in turn it’s biological function. If the structure of protein is modified by substitutions of important amino acid residues, our hypothesis is that its structure and function are most likely altered.To investigate this relationship, 25 single amino acid mutants of recombinant Taq polymerase gene were attempted using side directed mutagenesis. 23 of these mutants were successfully transformed into super competent cells. The wild type and all the mutant proteins were expresses and purified using ion-exchange chromatography. To observe the effect of mutation, we compare the melting behavior of the mutants with that of wild type protein. The protein denaturant guanidium hydrochloride was employed to generate denaturation profiles using circular dichroism spectroscopy to follow protein stability. 8 mutants have been analyzed so far.

Location

DeRosa University Center, Ballroom B

Start Date

1-5-2010 1:00 PM

End Date

1-5-2010 3:00 PM

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May 1st, 1:00 PM May 1st, 3:00 PM

Analysis of Structure-Function Relationship in Taq DNA polymerase

DeRosa University Center, Ballroom B

Taq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus. It is widely used in polymerase chain reaction (PCR). T. aquaticus was isolated from hot springs and Taq polymerase was identified as an enzyme able to withstand the thermal cycling conditions required for rapid amplification of DNA. Taq's optimum temperature for activity is 75-80°C, with a half-life of 9 minutes at 97.5°C.In current research, we used Taq polymerase to study the relationship between its structure and function based on the hypothesis that primary structure (amino acid sequence) codes for it’s folded 3D structure and in turn it’s biological function. If the structure of protein is modified by substitutions of important amino acid residues, our hypothesis is that its structure and function are most likely altered.To investigate this relationship, 25 single amino acid mutants of recombinant Taq polymerase gene were attempted using side directed mutagenesis. 23 of these mutants were successfully transformed into super competent cells. The wild type and all the mutant proteins were expresses and purified using ion-exchange chromatography. To observe the effect of mutation, we compare the melting behavior of the mutants with that of wild type protein. The protein denaturant guanidium hydrochloride was employed to generate denaturation profiles using circular dichroism spectroscopy to follow protein stability. 8 mutants have been analyzed so far.