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.
M L
ML
Kotewicz
J M
JM
D'Alessio
K M
KM
Driftmier
K P
KP
Blodgett
G F
GF
Gerard
D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D008970:Molecular Weight; D008979:Moloney murine leukemia virus / Q000235:genetics*; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*
D004274:DNA, Recombinant; D012194:RNA-Directed DNA Polymerase