TITLE: Recombinant Protein Expression of MMLV-RT H+ (SkiBar H+ RT) V2
AUTHORS: Alex Brown

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">About:</span></div><div class = "text-block">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.</div><div class = "text-block"><span style = "font-weight:bold;">Enzyme Engineering:</span></div><div class = "text-block"><span>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 </span><span style = "font-weight:bold;">enzyme contains an active RNAse H domain and is highly thermostable</span><span>. 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 </span><span style = "font-weight:bold;">Thermo Fisher Maxima H- RT</span><span>, 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.</span></div><div class = "text-block">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.</div><div class = "text-block"><span>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 </span><span style = "font-weight:bold;">H+</span><span> RT.</span></div><div class = "text-block"><span>Introducing mutations </span><span style = "font-weight:bold;">(D524G, E562Q, D583N and D653N) </span><span>and/or </span><span style = "font-weight:bold;">(D524, D583, E562, H204, V223, T306, F309)</span><span> are best suted eliminate RNase H activity completely, and it will increase the thermostability somewhat as well</span></div><div class = "text-block"><span style = "font-weight:bold;">Comments:</span></div><div class = "text-block">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</div><div class = "text-block">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.</div><div class = "text-block"><span>The plasmid used can be found on reclone.org and </span><a href="https://www.addgene.org/153312/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">addgene</span></a></div><div class = "text-block">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.</div><div class = "text-block">Tgatcc motif at MCS is a result of issues cloning in with BamHI. Had to use a compatible sticky end from BclI-HF</div><div class = "text-block"><span style = "font-weight:bold;">Protein Properties: </span></div><div class = "text-block">79.9 kDa</div><div class = "text-block">Isoelectric point (pI) : 7.77</div><div class = "text-block">Charge at pH 7.0: 9.52</div><div class = "text-block"><span style = "font-weight:bold;">Name:</span></div><div class = "text-block"><span>The name </span><span style = "font-weight:bold;">SkiBar H+ RT</span><span> is an abbreviation of the first two first authors where the MMLV mutations used in the construct were originally discovered (</span><span style = "font-weight:bold;">Ski</span><span>rgaila & </span><span style = "font-weight:bold;">Bar</span><span>anauskas).</span></div><div class = "text-block"><span style = "font-weight:bold;">References:</span></div><div class = "text-block"><span style = "font-weight:bold;">Bar</span><span>anauskas, Aurimas Paliksa, Sigitas Alzbutas, Gediminas Vaitkevicius, Mindaugas Lubiene, Judita Letukiene, Virginija</span></div><div class = "text-block">Burinskas, Sigitas Sasnauskas, Giedrius Skirgaila, Remigijus</div><div class = "text-block">Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants</div><div class = "text-block">Protein Engineering, Design and Selection (2012)</div><div class = "text-block"><span style = "font-weight:bold;">Ski</span><span>rgaila, Remigijus Pudzaitis, Vaidas Paliksa, Sigitas Vaitkevicius, Mindaugas Janulaitis, Arvydas</span></div><div class = "text-block">Compartmentalization of destabilized enzyme-mRNA-ribosome complexes generated by ribosome display: A novel tool for the directed evolution of enzymes</div><div class = "text-block">Protein Engineering, Design and Selection (2013)</div></div>

[STEPS]
?. [Protein Expression]
Streak CarbrCamr LB plate supplemented with from frozen stock of Rosetta DE3 . Grow plate overnight at 37°C.
?. [Protein Expression]
Select single colony from overnight streak plate and inoculate 5mL of LB broth supplemented with Carb and Cam. Grow overnight at 37°C shaking at 250rpm.Prepare 500mL LB and sterilize by autoclave.Allow sterile LB to come to room temperature overnight.
?. [Protein Expression]
1 mL of this culture was used to inoculate 500 mLs of LB in the morning. 500mL LB broth is then supplemented with Carb and Cam.The large culture was incubated at 37°C in a shaker at 275 RPM.OD600 was monitored until it reached 0.8-1.0 and was induced with 0.5 mM IPTG.Monitor OD600 every 30-40 mins. As OD600 approaches 0.5 monitor evey 5-10 mins.Induction occurred at 18°C for 20 - 24 hours.
?. [Protein Expression]
Collect cells by centrifugation. Conditions as follows: 4°C, at 4,000 x g for 30min.Cells were harvested by centrifugation, washed once with 1X Lysis Buffer (20 mM Imidizole) then resuspended in 5 mL of ice cold 1X Lysis Buffer per gram of wet pellet.Lysozyme was added to a concentration of 1 mg/mL and lysed by sonication, with 15 cycles of 10 seconds on/30 seconds off in salted ice water.Sonicate more if the cells are not completely disrupted (Lysis is complete when the cloudy cell suspension becomes translucent. Avoid protein denaturation by frothing).Spin 30min 18000rpm 4°C. Separate soluble proteins (supernatant) from insoluble or inclusion bodies proteins (pellet). Use supernatant for next step. Keep sample of 40µl of supernatant for PAGE-SDS: soluble proteinsResuspend pellet in 5ml column buffer and keep sample of 40µl for PAGE-SDS: insoluble proteins, or unlysed cells.
?. [IMAC Purification]
The following steps are performed at 4°CClarified lysate was then loaded onto 0.5 mL of Ni Sepharose Fast Flow beads (use whatever you got) that had been equilibrated with 1X Lysis Buffer (20 mM Imidazole).Collected flow-through was re-loaded onto the column and ran a second timeColumn was washed with 10 column volumes of buffer CEluted with 5mL Buffer D (500mM Imidazole)Each 1ml of the eleution buffer is collected separately in 1.5ml tubes.Eluted fractions are measured for protein absorbance on a nanodrop, using the imidizole containing elution buffer as a blank reference sample.samples values are recorded. Protein absorbance reads will peak and wane to background levels. Once eluted fractions are at background levels The column was regenerated with 10 mL washes of water/1M NaOH/water/50 mM EDTA/water/100 mM NiCl/20% EtOH and stored with 20% EtOH in the fridge.
?. [Amicon Size Separation or Dialysis]
Amicon protein concentrators can be substituted with dialysis into 50% glycerol containing storage buffer. Amicon protein concentration can cause proteins to crash out of solution if the solution is too concentrated, resulting in loss of protein yield. However they are much quicker than dialysis and do not require making large volumes of buffers containing 50% glycerol. Dialysis will minimize yield loss, but will not purify your protein from possible low molecular weight contaminates. Refer to ThemoFisher dialysis methods for implementing this approach.Separate protein using an amicon with molecular weight cut off ~1/3 the molecular wight of the purified target protein. MMLV = 79.8kDa, Amicon filter cutoff 25kDa.Exchange buffer with storage buffer at this point. Exchange with storage buffer containing 2× concentration of salts and no glycerol. Exchange buffer 3x, spin at 6000g 30-60 min each time, or until the
?. [Storage]
If using Amicon size separation protein can be stored in a storage buffer containing 50% glycerol. The exchange buffer contains 2× the concentration of salts and can be diluted 50% via the addition of 100% glycrol. From here aliquats can be mae and stored at -20C