Recombinant Protein Expression of MMLV-RT H+ (SkiBar H+ RT) V2 v2

Abstract

This protocol has been optimized for recombinant expression of molony virus based reverse transcriptases (RT). The plasmid used contains a reverse transcriptase gene which is derived from Moloney Murine Leukemia Virus. The enzyme when expressed recombinantly can synthesizes a complementary DNA strand from single-stranded RNA, DNA, or an RNA:DNA hybrid. Enzyme Engi ne e ri ng: Enzyme Engi ne e ri ng: The enzyme contains point mutations to generate a highly processive, thermostable, and improved fidelity mutant reverse transcriptase. Details, incluiding the sequence of this enzyme are published and can be found in the references below and addgene link. The e nzyme conta i ns a n a cti ve RNAse H doma i n a nd i s hi ghl y e nzyme conta i ns a n a cti ve RNAse H doma i n a nd i s hi ghl y the rmosta bl e the rmosta bl e. This MMLV construct contains the following mutations: D200N, L603W, T330P, L139P, E607K,D524G, E562Q, D583N and D653N. These appear to be the same set of mutations present in the T he rmo T he rmo Fi she r Ma xi ma H-RT Fi she r Ma xi ma H-RT , which is one of the best preforming RTs you can buy. The enzyme in our hands is extremely active. Moreover, while the MMLV RT is thermostable and will work up to 65°C it runs optimally at 42°C. The point muitations utilized here are currently filed under a provisional patent with Thermo Fisher Scientific. Commcercial use of this enzyme must go though Thermo Fisher Scientific.

Homebrew reagents for low-cost RT-LAMP

“…BstLF and M-MLV were produced from isopropyl b-D-1-thiogalactopyranoside inducible plasmids pET15b BstLF and pET-19b_MMLV-RT bearing 63 and 103 His-tags at the N terminus, respectively, and expressed in E. coli, according to the open access protocols described in protocols.io. 33,40 Then, the purification was performed in one Ni-NTA chromatography step for BstLF and in 2 chromatography steps (Ni-NTA and Heparin) for M-MLV, resulting in highly purified proteins in 3-4d. Typical protein purification yields from 1 L cell culture correspond to 4 mg of M-MLV and 10 mg of BstLF.…”

Homebrew reagents for low cost RT-LAMP

“…BstLF and M-MLV were produced from IPTG inducible plasmids pET15b BstLF and pET-19b_MMLV-RT bearing 6x and 10x His-tags at the N-terminus, respectively, and expressed in E. coli, according to the open access protocols Rivera et al, 2020 33 and Rivera et al, 2020b 34 (modified from Brown, 2020 40 ). Then, the purification was performed in one Ni-NTA chromatography step for BstLF and in two chromatography steps (Ni-NTA and Heparin) for M-MLV, resulting in highly purified proteins in 3-4 days.…”