PRODUCTION AND CHARACTERIZATION OF A CYSTEINE(-) M-MLV REVERSE TRANSCRIPTASE

Abstract

The mesophilic reverse transcriptases of Moloney murine leukemia virus and avian myoblastosis virus are commonly used by genetic engineers for cDNA cloning and RT-PCR amplifications. Synthesizing full length, high quality cDNA, especially acquiring sequence from the 5' end of mRNA, can be difficult. Truncated cDNA can be caused by competing RNase H activity, enzymatic pausing, non-specific primer annealing or obstructing RNA secondary structure. Superscript II (Life Technologies, Inc.) is a genetically engineered version of Moloney murine leukemia virus-reverse transcriptase (MMLV-RT) which retains full DNA polymerase activity yet lacks Rnase H activity. This modification eliminates the problem associated with RNase H mediated degradation of RNA transcripts during first strand DNA synthesis. A modified version of this reverse transcriptase which could function at higher temperatures could potentially eliminate the problems of non-specific primer annealing and RNA secondary structure, and further promote full length cDNA synthesis. There are several approaches to increasing the thermostabitity of a proteins. One approach is to insert a stabilizing cross link by the addition of a genetically engineered dihistidine metal bridge. However, there are two caveats to this approach. Firstly, the insertion of histidines is often destabilizing and can negate the stabilization conferred by the metal bridge. Secondly, exposed cysteines must be removed to avoid metal mediated oxidation of their sulfhydryl groups and subsequent denaturation. Superscript II has eight cysteines which are modeled to be in the free reduced state. This paper describes the creation and characterization of a cysteine- less MMLV-RT mutant protein. The characterization of this protein is important for establishing the properties of the protein before the dihistidine bridge is added.