Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli

A pBR322-derived expression vector, plasmid pKD1, was constructed containing the strong leftward promoter (pL) of bacteriophage lambda, the ribosome-binding site (RBS) of the cII gene of lambda, and a unique downstream NdeI restriction site for construction of an ATG initiation codon. The section of the pol gene of Moloney murine leukemia virus (M-MLV) that codes for reverse transcriptase (RT) was cloned into the NdeI site of this vector generating the plasmid pRT103. Upon thermal induction, enzymatically active RT was expressed in Escherichia coli [pRT103]. The identity of this activity was confirmed by its template specificity and its sensitivity to inhibition by immunoglobulin G (IgG) prepared against authentic murine RT. RT represented 20% of the newly synthesized protein in these cells 20 min after induction.