Published: September 6th, 2018 Last Modified: April 21st, 2020
Purification of pfu-sso7d ended up being a stunningly popular article, only being beaten out by DIY decontamination mix (people really enjoy decon mixes, apparently). What it revealed was that there is a global demand by scientists who wish to purify their own enzymes to be used as tools in their labs. I’m glad that I’m not the only one who has adopted the DIY ethos.
Another detail that surfaced is that besides an enzyme useful for PCR, a reverse transcriptase (RT) was in equally high demand. This isn’t surprising when you consider that RTs are some of the most expensive enzymes for RNA biologists and if you’re between funding cycles, lost your funding or not in a central country it can be a barrier to entry/continuation in the field. So, you asked, and I have delivered: MashUp (MU)-RT, a custom, FeLV based RT available to academic users!
Before we get into MashUp-RT, let’s settle in with a bit of history of RTs and then specific developments of the art.
The first commercially available RTs in the 80s were Avian Myeloblastosis Virus (AMV) RT and Moloney Murine Leukemia Virus (MMLV) RT. Generally speaking MMLV was found to be superior due to being several times more accurate.
Invitrogen (then Life…now Thermofisher?) began its monopoly of the high end RT market with the advent of Superscript II, which was patented globally in the late 80s. Based purely on the patent, it appeared that SSII was MMLV with a C-terminal truncation to remove RNase-H activity. However, the SSIII patent implies that instead of a truncation, SSII is full length MMLV-RT that has the following mutations: D524G, D583N, and E562Q (Possibly because the inactive RNase-H domain is useful, perhaps it improves RNA binding). By abolishing the RNase-H activity you not only increase the yield of cDNA, but also increase the thermostability of the enzyme to a nice 42C. It is important to note that the patents for SSII have expired (or are very close to expiring) so the market has been flooded with SSII variants/clones at a lower price.
(Wild type MMLV-RT sequence for those playing along at home)
Superscript III was the next development, with mutations that increase fidelity, thermostability and reduced terminal transferase activity: H204R, T306K, F309N, V223H, D524G, D583N, and E562Q. Overall, a very nice enzyme and one we’ve used until recently (I think SSIV is the same price, may as well buy that), you can do RT reactions at 50C comfortably, which allow you to use longer, more specific primers. As well, the higher temperature irons out secondary structure with can impede cDNA synthesis.
Stratagene (now Agilent) jumped into the game in 2005 with their patent that has the following mutations: E69K, E302R, W313F, L435G, N454K, D524N. Mutation at D524 knocks out RNaseH activity, E302 is mutated as in superscript IV (Below).
Applied Biosystems (Now owned by Thermo as of…?) also threw in their hat with:
F155Y, R301L, F309A, D524E or N, E562D, Y586A,H638G, D653N
Fermentas (Owned by Thermo as of 2010) patented a slew of mutations in MMLV in 2008:
E5K; M39V or L; I49V or T; M66L; Q91R or L; P130S; L139P; I179T or V; D200N, A or G; Q221R; Q237R; T287A; A307V; T330P; L333Q; Y344H; A502V; D524A; L528I; H594R, K or Q; L603W or M; E607K, G or A; H634Y; A644V or T; N649S; D653G, A, H or V; K658R or Q; and L671P.
How can they patent that many mutations? An interesting legal question is at what point is a molecular biology patent considered to be too broad?
Likely, the acquisition of Fermentas by Thermo laid the groundwork for the Superscript IV patent in 2014:
P51L, S67R, E69K, T197A, H204R, E302K, F309N, W313F, T330P, L435G, N454K, D524G, D583N, H594Q, D653N, and L671P.
(Mut D9 Sequence for those playing along at home)
This is one beauty of an enzyme, it can do 60C and is resistant to a number of inhibitors, likely the best commercial RT out now.
There are a few other patent holders in the art:
NEB recently filed a patent for a MMLV mutant, they do not disclose the exact mutations but instead claim a specific sequence. When aligned with MMLV, it has:
H8Y, S56A, T246E, N249D, Q291I, M320L, T330E
After amino acid 330ish there are no mutations, not even D524, not sure why. NEB could be a serious competitor to Thermo if they could provide a RT with similar performance to SSIV at a more digestible price.
A Japanese group published and patented MM4-RT, which has:
E286R, E302K, L435R, D524A
We’ve seen E302K and D524A before.
Finally, we have MMLV/FeLV domain chimeras, patented by Bio-rad, not sure exactly where they’re planning to take this, and why it’s superior to single domain RTs, but here we are. They only included D524.
I’ve compiled an excel sheet with all the mutations I could dig up compared against each other for your viewing pleasure, here.
So, what do we get from all this?
- There are a few hotspots within MMLV that significantly increase thermostability, fidelity and processivity.
- Patents owned by several different companies/individuals possess either identical amino acid mutations or mutations at the same position with amino acid changes that produce the same effect.
- ThermoFisher is the dominant player in the field, they’ve been doing it the longest and have acquired all the juicy mutations in MMLV
From what I can gather from the literature, the next movement in the field will be domain chimeras of different RTs (Bio-rad), fusing domains akin to sso7d to RTs, or using RTs from more diverse viruses than AMV or MMLV.
Now, all this research was used as inspiration to design MashUp-RT. This enzyme is based on FeLV-RT, which is intrinsically more accurate than MMLV. As well, it has mutations which increase characteristics like thermostability, accuracy, etc. Like pfu-sso7d, It should be a general workhorse enzyme for molecular biologists.
Now, It will take probably 2-4 weeks to fully characterize the activity of MashUp and to provide a comprehensive purification protocol, but those who are willing to participate in beta testing can contact me for the clone.
NOTE: Feedback for Mashup has come in, new users may want to read people’s protocols rather than follow what I have below. I’ve kept it so I can see how far we’ve come. According to Ryan, looks like you need a chaperone plasmid to make Mashup work with autoinduction media.
NOTE 2: Here’s our take on the Mashup purification and buffer optimization!
NOTE 3: Abridged, most up to date protocol: PDF, DOCX
Update #1 (27/9/18)
Thank you for all the beta testers coming forward, keep it coming! Just so everyone doesn’t start from scratch, here’s how the purification is coming thus far. I have purified MMLV RT before, and I used the following paper as a guideline: https://www.sciencedirect.com/science/article/pii/S016816561001895X
Unlike a standard protein purification, do not induce the expression of Mashup at 37C. Induce at 22-30C. I’m not a protein purification buff, but for many proteins the expression is drastically improved when done at a lower temperature. You can try both low and high temp, however my early results suggest that 37C does not work well, if at all. If you’re feeling fancy you can probably induce at 16C. Why does this phenomenon happen? Well, at higher temps protein synthesis is increased, sure, but a sudden high concentration of protein can induce aggregation into inclusion bodies or misfolding. As a side note, I am using Rosetta 2 DE3 pLysS cells for the purification. There should be no need for the rare codons the strain produces due to codon optimization of mashup, but it’s what I had in the freezer and the second antibiotic is nice to have.
My first attempt at purifying Mashup was by culturing in autoinduction media (AIM). Basically, instead of taking samples at regular intervals, waiting for the OD of the culture to get up to 0.5-1.0, adding IPTG etc, you simply add your starter culture/colony/glycerol stock straight to the AIM and let it grow overnight (16ish hours, into stationary phase). You harvest your cells in the morning and proceed with the usual his tag purification, lets look at the recipe and see how it works:
Per liter:
6g Na2HPO4
3g KH2PO4
20 g Tryptone
5g Yeast Extract
5g NaCl
0.5g MgSO4
6mL Glycerol OR 10 mL 60% Glycerol (Filtered) ***
2g Lactose OR 25 mL 8% Lactose ***
0.5g Glucose OR 5 mL 10% Glucose ***
(Allegedly you can substitute galactose for lactose, haven’t tried to confirm)
***Filter sterilize and add to autoclaved salts/tryptone, or mix up a big batch of everything and filter it all ***
So, your standard rich media with some buffering capacity and a bit of Mg for flavor. The sugars are what make it special. Your E.coli eats up the glycerol and glucose first and reach a high cell density. After that’s all used up, all that’s left is lactose, which when metabolized induces the expression of your plasmid instead of IPTG. Generally speaking AIM has worked well in our hands, and it certainly works well for purification of RTs
Update #2 02/10/18
Folks, it actually works, what I managed to purify works as an RT! I’m putting the mashup against superscript 4, the current top dog of the RT world, full comparisons coming ASAP, just so happy I didn’t design a bunch of garbage! Woop!
Update #3 05/10/18
Alright guys, important purification updates, auto-induction media is easy to use, but perhaps it isn’t the best fit for Mashup, a 200 mL volume did produce a fair yield of enzyme however not the amount I had expected. I autoinduced at 28C for 18 hours, re-suspended in lysis buffer (50 mM Sodium Phosphate pH 8, 500 mM NaCl, 10 mM Imidizole, 5% Glycerol) lysed with a sonicator (5 x 10s bursts, 30s rest) and put through 8 mL of Ni-NTA resin. Eluted with a manual gradient of imidizole, from 10 mM to 1000 mM. Feel free to jump in with protein purification advice!
So, I am definitely purifying Mashup, but strangely it is not a single band like you’d expect. Not a protein purification buff, but could it be degradation? Something funny due to the auto-induction media? Not saturating the column due to low induction? Not sure, either way I took the best looking fractions and concentrated it down in a 50 kDa millipore concentration column and used that in comparison to superscript 4. I didn’t do a bradford to measure protein content, at this point I was just praying that the enzyme was active. I did a RT-PCR comparing 20 units (1:10 dilution) of SSIV vs a 1/8th dilution of the concentrated mashup for a 400 bp chunk of mRNA starting with 1 ug total RNA (RT performed at 50C). PCR was performed with homemade pfu-sso7d.
Woohoo! It actually works! Now, I was not discouraged by the band intensity tapering off with increasing anneal temp. Why could that be? It suggests that the Mashup produced less cDNA, which in turn could be due to a lower concentration of Mashup vs SSIV. As well, SSIV uses a proprietary buffer (5 mL for 250$, seriously Thermo?). It’s possible that the standard 5X First Strand buffer that’s been used for the last three decades inhibits PCR, more on RT buffers later. I took the Mashup I had and further concentrated down to do a proper gradient vs SSIV. Same conditions as last time except I used fewer PCR cycles (30 vs 35) to try and tease apart starting cDNA amount.
Still works! Whew! I arbitrarily set the concentration of Mashup to 200 AU (so I wouldn’t have to do more math). I’m getting close to the concentration of SSIV, and at full strength, at a sample size of one, Mashup seems to produce more cDNA. Sorry for the hastily done nature of these gels, I wouldn’t call them quantitative or definitive just yet. I would say that Mashup is still not as concentrated as I would like, ideally the gradient would look similar between SSIV and Mashup. What’s interesting is that SSIV produced a non-specific band while Mashup did not! Very encouraging!
The different performance could also be due to difference in buffer compositions. I highly suspect that the RT buffer for SSIV has additives like trehelose or betaine. Will try RT-PCR with Mashup and SSIV buffer and see what happens. Buying SSIV buffer to use with Mashup isn’t really viable though due to the ludicrous cost (unlike homebrew pfu-sso7d and NEB’s buffer pack, 5 mL for 25$) .
Not going to continue with this specific prep of Mashup, it’s served its purpose as proof that the enzyme works, however the starting quality of the protein is average at best, probably due to the autoinduction media. Will be trying the classic IPTG induction, and I’ll actually do a bradford, whatever next!
Update #4 02/11/18
Crunching along, taking longer than expected, but I should know by now not to be too optimistic! With that said, some improvements with the purification. First, I auto-induced at 22C vs 30C. Also, I used Sigma’s cell lysis reagent B vs sonication, not sure what did it but take a look:
So, I’m still not saturating the nickle column but again, considering I had 5 mL of the last fraction I think the yield was workable. I did IPTG induction with 400 uM IPTG and it performed worse than autoinduction media.
Unfortunately, I think the Millipore 50 kDa columns I’m using for concentration is actually hurting my yields significantly, because after concentrating that last fraction I’m left with 5-10% of what I started with. Sad. Will be switching to PES columns (Millipore’s are regenerated cellulose)
So, assuming I can improve induction I think we’re almost there. Next batch is growing at 16C for >24 hours, as suggested by a reader 🙂 As well, including a heparin cleanup column after nickle purification.
After that, I will escalate to expressing chaperone proteins with the RT, as suggested by another reader. What’s plan C? Sub-cloning into a cold expression vector.
Thank you to everyone who is sending in advice, it means a lot to me!
Update #5 13/11/18:
At the moment auto-induction at ~16C for 30 hours is looking the best so far, even taking into account that the incubator dropped to 10C for an unknown amount of time.
Expression levels are increasing! Not quite a slam dunk, perhaps due to the incubator getting too cold, but miles ahead of the last few attempts. Concentrated protein is active, however I’ll save full validation of activity for when I purify the final batch.
At this point I’ve decided to try and push expression levels a bit higher by sub-cloning into a cold-inducible vector as well as expressing some chaperone proteins. This will take a bit of workup but we’ll get there in the end!
Based on the SDS-PAGE results, I think degradation of the Mashup RT occured during lysis and purification. Would you try to add some protease inhibitors to the buffers?
Thanks for the advice, I actually did add a bit to the lysis buffer, but I guess I was being a bit cheap and didn’t add enough. I’ll try more next time. Perhaps the sonication was too much? I had the tube submerged in salty ice water so it kept it nice and cool. Either that, or I need to induce at 16C, will be doing that too.
Can you send me sequence for superscript IV , am confused reg mutation in table and patent . you said it has deletion also
Great article. I am a protein biochemist by trade, so I can offer some insight into your preparation.
First, autoinduction media works by depleting alternative carbon sources before inducing with lactose. All the other components are present to maintain the media pH and to provide trace nutrients that rich media typically lacks. Generally this won’t make much of a difference, although for polymerases supplementing with metals is a good idea. You may be cutting your induction short, since cells need to exhaust their supply of glucose and glycerol before they begin to “auto induce” with lactose. Typically we use 24 hours as our endpoint but you may not see an improvement in yield. Some proteins really do express better using IPTG.
Breakdown is likely what you see in your purification figure but since you only purified by NiNTA a fair amount of contamination (say 5%) is entirely expected. If you want to add a second purification step using a heparin resin is a good idea. These are unfortunately very expensive but heparin resembles the backbone structure of nucleic acids and so nucleic acid binding proteins stick very well to these resins. You elute with salt as with ion exchange resins. Judging from your gel you would likely have a high purity protein after this step. It should also have enhanced activity since non-binding protein will not stick to the column.
I stumbled on this article since I may be generating RT for a laboratory I just joined (today!). Have you seen Elefson et al. 2016 in Science? It may be relevant to this here.
This is good stuff! I may be producing RT for an upcoming position if I do well at the interview on Monday. Might save the company a bunch of money if it works as well as their current standard. We’ll see if they hire me! haha. I love this site.
Good luck bro, hope you get the job!
This is golden, thank you. Joel, is the RTX plasmid available? What a great article from Elefson et al. 2016.
This is very cool. I may be trying something similar. This is the input that I can provide as a protein biochemist:
1) PMSF is an inexpensive *irreversible* protease inhibitor with broad specificity for serine and cysteine proteases. Generally speaking, those are the ones you’re trying to hit. Since polymerases (e.g. reverse transcriptases) require metal cofactors you obviously can’t include EDTA in your lysis buffer.
2) Even if you throw every protease inhibitor known you are still going to get proteolysis, especially for larger proteins. Your purity is actually pretty reasonable for a single Ni-NTA column. If you want to get very high purity enzyme you should include a heparin affinity column step. Heparin vaguely resembles the structure of a nucleic acid backbone and works surprisingly well for the purification of nucleic acid binding proteins including polymerases. Unfortunately heparin columns are expensive ($40/ml of resin) but they are absolutely worth it for these purposes. Heparin has weak cation exchange properties, on the level of CM if not weaker, but typically you would not use it for that purpose.
3) Awesome job scouring the patents for this and the decon solutions. I once did the same for cell lysis reagents (BPER and Bugbuster) to try and improve solubility of a lipid-bound protein (it did not work). The secret ingredient in those is B-octylglucoside for what its worth, and it is extremely expensive.
4) Have you looked at Ellefson et al. (2016) in Science? They engineered a reverse transcriptase from a proofreading KOD polymerase. The result is a proofreading reverse transcriptase with high fidelity and thermostability. The plasmids are available from Addgene. I will definitely attempt to generate this enzyme.
Thank you so much for your input, I am not a protein guru by any means so it’s appreciated! 🙂 I’ll shoot you an email so we can talk some more shop!
Yes, you are probably right about the auto-induction not going to completion, I was following the protocol of MM4 RT purification, which did 30C for 16 hours. My batch growing at the moment is at 16C, and I’m gonna let it go for over 24 hours, so we’ll see. I’ll dig around in the fridge and see if we have some heparin and give your suggestion a go. I think I need to accept that RTs are a bit more tricky to purify than pfu-sso7d which was a piece of cake, expecting 95% pure protein after a single nickle column is pretty optimistic!
Also, I’m using millipore 50 kDa cutoff columns for concentration, I think I’m losing most of my protein on it, considering I had 5-10 mL of diluted mashup, and after the column it looked like I had even less. Those columns are regenerated cellulose, ordered some PES columns from sartorius, will see how that works.
Yes, I have looked into that science paper, as well as MarathonRT. I decided to go for the classic retroviral RT so that people could use it as a drop in replacement for existing protocols, from a cursory glace it seems like there is usually some sort of drawback in exchange for increased fidelity/processivity of these new RTs.
I think a 50Kda MWCO might be a bit too large. Try 10kda or so. the rule of thumb is a MWCO of a third or less of the mw of your protein. You can really push this to 2/3 (so a 100Kda membrane will work for a 150Kda IgG for example) but generally speaking it impacts yield. less than 50% recovery suggests either it binds to the membrane (unlikely with regen cellulose but maybe add 0.01% Tween20?) or its going straight through. Remember, the 50Kda MWCO is the average. some pores will be 80Kda and some will be 30 Kda etc etc.
Thank you for your insight on purification columns, I really appreciate it! I will do as you suggest on my next attempt. Several labs who asked to beta test have been optimizing the Mashup expression and are having success, and it’s clear I’ve been overthinking the expression and purification. I’m going to post a follow up post with their protocols and my own attempts soon, just been swamped with my PhD work!
I don’t know the prices around where you live but I have purified helicases for my PhD before and we didn’t had heparin columns. What I did was after NiNTA, dialyse into a low pH buffer (MES 5.5) and do an anion exchange (I had MonoS prep colums in the lab, really high resolution but may be quite pricey…) with respect to a KCl gradient, worked great! Then I got heparin columns and my procedure was superior (for DEAD box helicases anyway).
And usually regular IPTG induction is superior to autoinduction, but I would recommend using different media and see which one is better in protein expression for your specific protein. In my PhD lab, everybody was using Terrific Broth, but for my protein LB was much better than TB. This is the magic part of molecular biology and biochem – sometimes inferior stuff works better 🙂
I also added a PEI precipitation step (needs optimization though, for not to also precipitate your protein, which can be bound to non-specific ss or ds DNA) to clean contaminating DNA since you would not want DNAse treatment.
Also if you are aiming for cold expression, you can go with ArcticExpress cells or get Takara’s Cold-inducible chaperones which greatly aid in production of soluble protein. Also use a cold shock promoter vector for your protein expression (like CspA promoter). You can go down to 8 degrees celcius, 48 hour expression post induction.
And I have a comment for this “Since polymerases (e.g. reverse transcriptases) require metal cofactors you obviously can’t include EDTA in your lysis buffer.” Most of the time what divalent cations do is to associate with ATP to form ATP-Mg (for instance) that attach to Walker A motifs. If there is no structural divalent cations in your protein, you can EDTA the shit out of this and dialyse the shit out of EDTA when you are finished. You can always add a pinch more of the Mg during the experiment.
I am impress with your expression data. I feel like you can get much better purification if you lower the amount of NiNTA used. I would recommend 1/10 the amount of resin or even lower for the amount of protein you are getting from your purification. I have always found that lowering the amount of NiNTA improved the overall quality of the purified protein.
Thank you so much for your input, I was thinking something like that was happening, I will give it a try! I’m not an expert with protein purification, so you guys will have to bear with me 🙂
Shell we have the DNA sequence for the Mashup-RT 🙂
Thanks
Hey, at the moment I’m not publicly releasing the DNA/amino acid sequence of the RT on my blog, but those who request the plasmid can certainly have it. I’ll shoot you an email.
Thanks for sharing the vector.
We have got not bad yield (0.2 mg / 50 ml culture) using BL21 induced with 0.8 mM IPTG at 16C for 24 hours, one-step NiNTA. We found the prep was inhibited by the buffer from the ABI kit, but the prep works well in universal buffer (Tris-HCl 8.3 50 mM, KCl 75 mM, MgCl2 3 mM and 10 mM DTT). All first strand reactions were done at 37C for 2 hours, and not much change at 50C.
We also run qPCR with two housekeeping gene primers and two single copy gene primers. For housekeeping gene, the Cq from homemade buffer/MachUp is ~ 2 larger than from the ABI kit(like 20 vs 18). For single copy gene, the homemade one always got lucky—- smaller Cq (like 35 vs. 37).
We didn’t check the RT concentration in the kit. We always use 0.05 mg/ml for MashUp.
Any idea for improvement?
Thank you for sharing your experience, you are the first one to come back with data, you’ve made me so happy!
So, I think that the main improvements are going to come with optimization of the buffer. High end commercial RTs like superscript add various additives to their RT buffer which improve their performance, just judging from the SSIV patent (https://patents.google.com/patent/US20180010105) it looks like 10-15% trehalose and 0.05 to 0.1% detergents/surfactants.
Here’s another paper regarding trehalose: http://sky.njnu.edu.cn/sites/default/files/inline/files/dengcheng_article_9.pdf
So yeah, there’s a good bit of optimization reactions to be run, but your early results are encouraging!
I’m currently working on improving the yield by sub-cloning into a cold inducible vector, along with co-expressing chaperone proteins. I’ll let you know how it goes. Thank you again.
Thank you for suggestions. I will definitely try trehalose and detergent. Do you have any idea about the processivity of MashUp compare to MMLV?
Our ArcticExpess seems not growing in newly bought Tetracycline…… FYI, we used 60 mM imidazole to wash the column or gradient elution to get rid of chaperon from NiNTA 🙂
I am quite sure that I have made a looooong comment to here… Bah, whatever 🙂
I used to purify a lot of viral helicases during my PhD. One thing I used instead of a heparin column was to use an S (strong anion exchange) column. What I did was (for HCV helicase) to lower the pH after NiNTA to 5, lower salt to 5o mM, apply that to a MonoS HiLoad. Quite pricey columns but quite nice.
Nevertheless, one thing I always did with DNA-binding proteins was immediately to pass them through a Q (strong cation exchange) column to get rid of DNA. Excess DNA sometimes is known to act as a competitive inhibitor for DNA binding proteins so you can either precipitate them with PEI (not the variant you use for transfection, I’ll look through which one we used) or pass it through a DEAE at the simplest. Also it is a nice clean-up step, since DNA proteins can tolerate to a shit ton of salt (I saw 1.6M before my protein crashed). You can dislodge DNA/protein interactions and by careful optimization your DNA gets stuck to the column while your protein flows freely.
Since we purified also the HCV NS3/4A protease/helicase complex, we could not use protease inhibitors but I have never encountered significant degradation in my protein. It is useful to err on the cautious side though…
Lastly, TaKaRa had a cold inducible expression system with trigger factor and chaperone plasmid set. If you have them, use it and do your expression at 8-12 degrees, 48 hours. SAVED MY PHD – NOT JOKING.
Hello! It was interesting to read about the work you do. A couple of years ago, I synthesized revertase with mutations from ssII, agilent and fermentas patents, which I successfully get and use in my work. I can advise you not to use autoinduction during MMLV expression. In my experience, MMLV is 70-90% in the inclusion bodies, and induction using lactose (galactose) promotes their formation. I conduct induction in standard BL without plysS plasmid, since MMLV is not very toxic to cells. I use LB + glucose (1%) for catabolite repression of promoter leakage. The cells are grown at 37 ° C to an optical density of 1, then I reduce the temperature to 25 ° C and bring IPTG to 0.2 mM. Induction takes 5 hours. On average, from 1 liter of culture, 5 mg of purified MMLV is obtained (Ni-IDA plus desulting) with an activity of about 200,000 U / mg.
Wonderful information, thank you comrade! I’ll shoot you an email so I can ask you a few questions.
it’s very nice to read such an interesting knowledge share. thanks so much!
but your protein expression and purification procedures need some modifications.
enzyme purity determines its sensitity and stability, and expression amount affects homemade reagent’s cost.
good luck.
Glad you enjoyed the read, and thank you for the encouraging words.
I’m still actively working on the purification, and there’s still a long way to go, but I intend to see this through. I want to see it at the same level as homemade pfu-sso7d, with a simple purification protocol and high activity.
anyone tried activity in inclusionbody RT expression
Great work. I tried DIY MutD9 at my lab, got bucket loads of it with just 100 mL Culture in Auto induction expression.
I’d say coexpress with Chaperone plasmid definitely would help. Let me know if you need any chaperone plasmids, happy to send them your way if you are not too far from Australia.
Glad to hear you found the post useful 🙂 Sent you an email so we can talk shop.
My good friend!
Thank you that you continue to work on Mashup-RT!
I would also like to help a little, I recently came across a very interesting article:
An ultraprocessive, accurate reverse transcriptase encoded by a metazoan group II intron. Zhao C, Liu F, Pyle AM. RNA. 2018 Feb; 24 (2): 183-195. doi: 10.1261 / rna.063479.117
Particularly pleased that the plasmid is available on Addgene:
https://www.addgene.org/109029/
Thanks man, hope you’re doing good! Yeah the feedback for MashupRT has been really good, take a look at the update post if you are going to be doing a purification soon https://pipettejockey.com/2019/04/19/mashup-rt-beta-update-1/
Yes, we’ve actually requested MarathonRT before, however I think it got stuck at the stage of MTAs etc.
Now that I know we can get it off Addgene that changes things!
I followed the expression tricks in feedback got about 20 mg / liter culture after elute from Ni-NTA. However, after dialysis to the storage buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.01% (v/v) NP-40, 50% (v/v) glycerol), about half of the RT precipitated out. The final concentration is 1.2 mg/ml. We don’t have suitable set for testing the unit. So we just did a roughly quantification. I hope this data could help people cannot do unit defination. By comparing a commercial MMLV RT kit, first strand synthesis are done at 37C 2hours and followed qPCR using beta-actin and GADPH primers.
Commercial 60xRT 180xRT 400xRT 540xRT 1680xRT
beta 17.2 16.78 16.51 16.81 17.04 18.32
GAPDH 18.22 17.76 17.77 17.58 17.69 18.96
Also, we tested the one-step qPCR mix using taqman primers, the starting buffer is from here, 5X Mashup Buffer (5X MB)
(mM) 80 70 60 50 40
KCl 25.80 25.81 25.91 26.1 26.6
(mM) 1.5 2 2.5 3
MgCl2 28.5 25.91 25.42 25.19
50x 200x 400x 800x
RT – 25.91 25.4 26.6
So our final buffer for one-step qPCR is
4x one-step qPCR buffer
100 mM Tris-HCl pH 8.3
280 mM KCl
10 mM MgCl2
25% Glycerol
0.03% NP-40
100x RT (1.2mg/ml stock)
200 uM each dNTP
1000x e.coli UNG (0.2 mg/ml stock)
100x Taq pol (1 mg/ml stock)
Increasing MgCl2 to 3mM gives smaller Cq, but the amplification curve looks not good.
Also adding 3% DMSO could prohibit false positive for some primer set, and push the Cq back 0.5 more. I think it is still worth to add.
Thank you for the wonderful feedback Ye, I’ll put it into the second post in the contributor section 🙂
Hello Yang
We too see lots of precipitation once Ni elutes are dialysed in storage buffers. Tried with varying detergents/concentrations but still precipitation is there.
If you have identified some root cause pls pls let me know
I think that Mashup can bind a lot of nucleic acids during lysis and pull down, one researcher does an on column wash of wash buffer plus NaCl to wash off the nucleic acids.
Hi Alex,
Could I please they the vector maps for the pML-MashupRT and pML-MRI (legacy vector)?
Thank you,
Yogesh
We are trying to use MashUp to do a long range RT. Seems not working. Just curious, why FeLV was used here. Because higher fidelity? I found a set of data for FeLV and MMLV, for FeLV, steady state kinetic analysis, for dNTP average Km is ~2 uM and Kcat is ~0.2, meanwhile for MMLV, pre-steady state kinetics analysis, for dNTP average Km is ~10 uM and Kcat is ~60. So the Kcat/Km is 0.1 vs 6. Despite pre-steady data usually is better than the steady state, still, it looks lot differences. Seems like the FeLV is not that efficiency compare to MMLV (because high fidelity? like pfu pol vs. taq pol). Do you have more papers about FeLV used in routine RT reactions?
Hi!
It is very interesting article!!! For past 2 years our lab was trying to purify the MarathonRT but with no success. So have been looking for alternative and very pleased to find such detailed protocol. Awesome job!!!
Is it possible to get MushUp vector somehow?
Thank very much for your research, but i can’t find the superscript IV patent, I’ll very appreciate if you can send me this patent.
Hi, Thank you so much for sharing, your blog feels like I am in a new world! May I ask you the DNA sequence of Mashup-RT? could you drop me an email with Mashup-RT plasmid seq?
Thanks!