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Cloning, expression, and purification of a catalytic fragment of moloney murine leukemia virus reverse transcriptase: Crystallization of nucleic acid complexes
Author(s) -
Sun Dunming,
Jessen Sven,
Liu Chunhui,
Liu Xiuping,
Najmudin Shabir,
Georgiadis Millie M.
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560070711
Subject(s) - reverse transcriptase , dna polymerase , nucleic acid , microbiology and biotechnology , dna , polymerase , biology , dna polymerase i , murine leukemia virus , rna , rnase h , rna directed dna polymerase , biochemistry , chemistry , gene
Reverse transcriptase is an essential retroviral enzyme that uses RNA‐ and DNA‐directed DNA polymerase activities as well as an RNaseH activity to synthesize a double‐stranded DNA copy of the single‐stranded RNA genome. In an effort to obtain high‐resolution structural information regarding the polymerase active site of reverse transcriptase, we have pursued studies on a catalytic fragment from Moloney murine leukemia virus reverse transcriptase. DNA encoding the catalytic fragment, defined originally by limited proteolytic digestion, has been cloned, and the protein has been expressed and purified from Escherichia coli . The fragment obtained by limited proteolytic digestion and the bacterially expressed fragment retain polymerase activity. Crystallization studies involving nucleic acid complexes with a catalytic fragment from both sources are reported, including variables screened to improve crystals and cryocooling. Three crystal forms of catalytic fragment‐nucleic acid complexes have been characterized, which all contain at least two protein molecules in the asymmetric unit. As isolated, the catalytic fragment is monomeric. This analysis indicates that the enzyme dimerizes in the presence of nucleic acid.