FIV-FCA and FIV-PCO Reverse Transcriptases: Expression, Purification and Activity Comparison
Wang, Jingjing, October 2006
Feline immunodeficiency virus from domestic cats (FIV-Fca) and cougars (FIV-Pco) are genetically related but they have significantly different evolutionary rates and virulence in their natural hosts. Additionally, recombination in FIV infested cats is common but there is no evidence for recombination in endemic cougar infections. The reverse transciptase (RT) encoded by FIV plays a key role in the virus life cycle and is central to both evolutionary rates and recombination events. Our hypothesis was that replicating enzymes from a virus with a long coevolutionary history with its host will have higher processivity. In this thesis we expressed, purified and compared the processivity of recombinant His-tagged RTs of FIV-Fca and FIV-Pco.
Both of the RTs contained a hexa N-terminal His tag and were expressed in E.coli. Both RT subunits were independently cloned and expressed but were isolated together by mixing induced cultures prior to affinity purification on a Ni2+ column.
The heterodimer conformation of the enzyme was confirmed by the gel filtration and SDS gel electrophoresis. Both of the RTs were highly active in an in vitro RT assay.
We compared the processivity of both RTs on an RNA template. However, no difference in processivity was detected under the conditions used in this assay.
Advisor:Dr. Mary Poss; Dr. Michelle McGuirl; Dr. Joh Gerdes
School:The University of Montana
School Location:USA - Montana
Source Type:Master's Thesis
Keywords:division of biological sciences
Date of Publication:03/02/2007