Recombinant Protein Expression of MMLV-RT H+ (SkiBar H+ RT) V2 v2
Authors: Brown Alex 
Published: July 2020

About: 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 Engineering: 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 enzyme contains an active RNAse H domain and is highly thermostable. 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 Thermo Fisher Maxima 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. It is also possible to further engineer this enzyme with to increase thermostability and eliminate RNase H activity to generate an enzyme which closely mimicks Thermo Fisher Maxima H+ RT. Introducing mutations (D524G, E562Q, D583N and D653N) and/or (D524, D583, E562, H204, V223, T306, F309) are best suted eliminate RNase H activity completely, and it will increase the thermostability somewhat as well Comments: Reference 2 highlights several point mutations which can be introduced to eliminate Rnase H activity which may also enhance RT sensitivity. Thermo Product information brochure from 2013 suggest that this mutant or some combintation of other identified point mutations from reference 2 is Maxima RT, Maxima H-. The paper has also identified a few more useful point mutations which may further optimise MMLV-RT This expression protocol can also implemented to express other MMLV based reverse transcriptases or diversely related retroviral RTs, provided the genes are in an expresison construct with an N- terminal 6-10 His Tag and are expressed under a T7 promoter. The plasmid used can be found on reclone.org and addgene SkiBar H+ RT was synthesized as a gBlock from IDT and is codon optimized. Expressed in Rosetta DE3 his tag is on still but seems to work fine with it on. Tgatcc motif at MCS is a result of issues cloning in with BamHI. Had to use a compatible sticky end from BclI-HF Protein Properties: 79.9 kDa Isoelectric point (pI) : 7.77 Charge at pH 7.0: 9.52 Name: The name SkiBar H+ RT is an abbreviation of the first two first authors where the MMLV mutations used in the construct were originally discovered (Skirgaila & Baranauskas). References: Baranauskas, Aurimas Paliksa, Sigitas Alzbutas, Gediminas Vaitkevicius, Mindaugas Lubiene, Judita Letukiene, Virginija Burinskas, Sigitas Sasnauskas, Giedrius Skirgaila, Remigijus Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants Protein Engineering, Design and Selection (2012) Skirgaila, Remigijus Pudzaitis, Vaidas Paliksa, Sigitas Vaitkevicius, Mindaugas Janulaitis, Arvydas Compartmentalization of destabilized enzyme-mRNA-ribosome complexes generated by ribosome display: A novel tool for the directed evolution of enzymes Protein Engineering, Design and Selection (2013)