An inverse polymerase chain reaction (PCR) was employed to construct an engineered F1-ATPase by means of inserting the repressor of primer (Rop) DNA sequence into the region of the ATP synthase gamma (γ) subunit DNA sequence encoding a regulatory dithiol-containing domain. A two-step PCR approach was developed to insert two unusually long (>100 base pairs each) primers encoding 189 base pairs of exogenous DNA into a single site within a pACYC multiple cloning host vector. The construct was verified by means of DNA sequencing. This approach allowed direct insertion of large pieces of DNA into a host DNA molecule without introducing restriction enzyme sites, thus avoiding common shortcomings such as inclusion or omission of base pairs that were associated with traditional sub-cloning methods. The engineered gamma subunit was designed for assembly with the recombinant alpha (α) and beta (β) subunits into a core F1-ATPase. The rigid twisted helical structure of the Rop protein extended the regulatory domain of the gamma subunit by approximately 60 Ångstroms, thus creating a rigid, rotating armature within the enzyme. The armature is intended for use as a site for attachment of gold particles to monitor rotation of the gamma subunit during ATP hydrolysis.
|Advisor:||Richter, Mark L.|
|Commitee:||Benedict, Stephen H., Davido, David O.|
|School:||University of Kansas|
|Department:||Biochemistry & Molecular Biology|
|School Location:||United States -- Kansas|
|Source:||MAI 47/06M, Masters Abstracts International|
|Keywords:||ATP synthase, Cloning, Gamma subunits, Insertion, Inverse PCR, Rotational kinetics|
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