Expression and purification of recombinant scaffold polymerase based on Φ29 DNA polymerase and terminal protein

Sethy, Sandeep Kumar (2023) Expression and purification of recombinant scaffold polymerase based on Φ29 DNA polymerase and terminal protein. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS dissertation of Sandeep Kumar Sethy (18MS160)) Thesis_18MS160.pdf - Submitted Version Restricted to Repository staff only Download (2MB)

Abstract

The DNA polymerase of bacteriophage Φ29 is a member of B-family polymerases. Φ29 DNA polymerase employs a terminal protein (TP) to initiate protein-primed replication. A specific serine residue in Φ29TP provides the hydroxyl group necessary for the first nucleotide incorporation. Several biochemical and crystallographic investigations indicate that interactions between certain Pol and TP domains help position the priming serine, S232, in the polymerase's active site for priming. Based on the co-crystal structure of Φ29 Pol and TP we designed two chimeric polymerase scaffolds that included substrate positioning function of TP to identify a construct capable of utilising a minimal TP or TP peptide as a protein primer. Expression and purification studies of both scaffolds were conducted. We report that the expression was poor, resulting in a meager yield after purification. Preliminary scaffold activity assays indicated that these proteins are active, however, due to a lack of pure protein, additional studies could not be completed within the duration.

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