Masters Theses

Date of Award

8-2001

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Elizabeth E. Howell

Committee Members

Barry Bruce, Steve Kennel

Abstract

Two clones have been constructed for the phage display of active R67 dihydrofolate reductase (DHFR). These clones will be used in fixture experiments for affinity selection of mutants with increased catalytic efficiency. R67 dihydrofolate reductase is an R-plasmid-encoded enzyme that confers clinical resistance to the antibacterial drug trimethoprim. The enzyme shows no sequence or structural homology to the Escherichia coli chromosomal DHFRs. R67 DHFR is a homotetramer with an active site pore that traverses through the length of the protein. The protein possesses exact 222 symmetry. The symmetry element allows for binding of one DHF or one NADPH molecule in each half-pore site although they are bound differently. Crystal structures with bound folate and NADP^ are not available for evaluation of R67 DHFR's hydride transfer reaction. Also, mutations in the active site inevitably produce four mutations per enzyme; this further complicates studies of its mechanism. Phage display of R67 DHFR will allow for selection of randomly mutated variants possessing increased catalytic properties that could be the result of a diverged active site or addition of a general acid. It is not likely that the phage display of the 78 amino acid monomer of R67 DHFR can work because of complications associated with the formation of the homotetramer on the surface of virions. Therefore, a quadruple R67 DHFR was used where four monomers are covalently linked and folds into an active enzyme fused to either gene III or gene VIII phage coat protein. This is the first case of a fission involving a tandem array. The first step leading to the phage display of the quadruple R67 DHFR was to construct two gene fusion constructs that contained the single gene copy of R67 DHFR linked in frame to the gene III or gene VIII of M13 bacteriophage. Then these two constructs were used as templates for engineering the two fusions containing the quadruple R67 DHFR fused to the gene III phage coat protein or to the gene VIII phage coat protein for phage display. These two fusions take advantage of the different display environments that each system has to offer. All four constructs conferred trimethoprim resistance upon host E. colt Purified phage indicated the quadruple R67 DHFR was displayed as an active en2yme. However, altered Km values were noticed. No exact assessment can be made for the altered Km values observed and the low activity measured. The altered properties of the fusion product do allow for a general trimethoprim resistance selection A more efficient selection will use a bisubstrate analog in future experiments.

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