Prokaryotic expression and purification of moloney murine leukemia virus reverse transcriptase and verification of the activity.
- Author:
Xiansong WANG
1
;
Xuemei MA
;
Yi SUN
Author Information
1. College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100022, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Mice;
Moloney murine leukemia virus;
enzymology;
genetics;
RNA-Directed DNA Polymerase;
biosynthesis;
genetics;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
metabolism;
Recombination, Genetic
- From:
Chinese Journal of Biotechnology
2008;24(5):903-906
- CountryChina
- Language:Chinese
-
Abstract:
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.
