A directed approach to improving the solubility of Moloney murine leukemia virus reverse transcriptase.
One of the difficulties that can impede structural work on a molecule of interest is limited solubility. Although functionally similar to the human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT), the Moloney murine leukemia virus reverse transcriptase (MMLV RT) differs both in architecture and solubility properties. Reverse transcriptase is an essential retroviral enzyme that replicates the single-stranded RNA genome of the retrovirus producing a double-stranded DNA copy, which is subsequently integrated into the host's genome. We have introduced a single amino acid substitution in the connection domain of an N-terminally truncated MMLV RT (L435K) that significantly improves the solubility of the enzyme eliminating the need for nonionic detergents in buffering storage solutions. The substituted enzyme retains near wild-type polymerase activity. An important consequence of the improved solubility of the L435K MMLV RT has been the ability to obtain diffraction quality crystals.
Das
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Waksman Institute and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, USA.
Georgiadis
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MM
D019943:Amino Acid Substitution; D003460:Crystallization; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D012995:Solubility
D012194:RNA-Directed DNA Polymerase