[Prokaryotic expression and purification of moloney murine leukemia virus reverse transcriptase and verification of the activity].
To produce the reverse transcriptase of moloney murine leukemia virus (MMLV-RT) through gene recombination, MMLV-rt gene was amplified by polymerase chain reaction (PCR) with specifically designed primers bearing restriction enzyme sites. Five mutation sites increasing the solution of the target protein were introduced through Site-directed mutation. After verification by sequencing, the gene was cloned into the expression vector pET15b to construct the recombinant plasmid pET15b-MMLV-rt. Purified MMLV-RT was obtained by affinity chromatography (Ni3+-NTA beads). Molecular weight and purity of MMLV-RT were analyzed with SDS-PAGE. Enzyme activity was characterized with RT-PCR. We successfully constructed the recombinant plasmid pET15b-MMLV-rt and obtained the MMLV-RT fusion protein with 6His on the N-terminus. Recombinant protein was purified through Ni3+-NTA beads based affinity chromatography, the purity of which was 96%. The Activity of the enzyme was high. MMLV-RT of 96% purity was obtained with the prokaryotic expression technique, which serves as the basis for mass production of this enzyme.
Wang
Xiansong
X
College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100022, China.
Ma
Xuemei
X
Sun
Yi
Y
D000818:Animals; D051379:Mice; D008979:Moloney murine leukemia virus / Q000201:enzymology* / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic
D011993:Recombinant Fusion Proteins; D012194:RNA-Directed DNA Polymerase