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

• Published in: Gene Vol. 35; no. 3; pp. 249 - 258
• Main Authors: Kotewicz, Michael L., D'Alessio, James M., Driftmier, Katharine M., Blodgett, Karen P., Gerard, Gary F.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 1985; Amsterdam Elsevier; New York, NY
• Subjects: active enzyme folding; Bacteriophage lambda - genetics; Biological and medical sciences; Biotechnology; Cloning, Molecular; DNA, Recombinant; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Genetic engineering; Genetic technics; Genetics; Methods. Procedures. Technologies; Microbiology; Molecular cloning; Molecular Weight; Moloney murine leukemia virus - genetics; phage λ vector; pL promoter; Protein Conformation; Recombinant DNA; reconstruction of the ATG codon; RNA-Directed DNA Polymerase - genetics; Virology; phage λ vector; reconstruction of the ATG codon; aa; active enzyme folding; p L promoter; DTT; M-MLV; buffer A; IgG; AMV; Recombinant DNA; bp; Ap; BSA; kb; EtBr; Oncovirinae; Temperature; Bactériophage lambda; Induction; Escherichia coli; Retroviridae; Gene expression; Immunological method; Virus; Lambda phage group; Gene; Styloviridae; Type C oncovirus; Substrate specificity; DNA polymerase RNA-dependent; Bacteria; Bactériophage; Murine leukemia virus; Molecular cloning; Escherichieae; Vector; Enterobacteriaceae
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(85)90003-4

A pBR322-derived expression vector, plasmid pKD1, was constructed containing the strong leftward promoter ( p p) of bacteriophage λ the ribosome-binding site (RBS) of the ell gene of λ, 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 m these cells 20 min after induction.