Published: August 14th, 2020 Last Modified: September 30th, 2021
Apologies for the hiatus and slow replies everyone, I’m entering one of the busiest parts of my life (to date…) and I will hopefully have a nice surprise update in a few months 😉 During these busy times I often turn to the lab bench as a sort of stress ball, and I figured it was time to share a few things I’ve been fidgeting with.
This release is probably the biggest to date with 4 new plasmids! My hope is that these enzymes will fill some holes in the current lineup. The ultimate goal is a small, curated pack of plasmids that are broadly applicable to molecular biologists.
1) Cheeky TEV (CTEV) – Everyone and their cat has the old-school TEV plasmids at this point, but there’s a whole world of TEV variants to explore and here’s my cheeky version of the current state of the art!
2)HIS-MBP-TEV-MRI and HIS-MBP-Sumo-MRI – My pML-MRI plasmid is being replaced by two superior versions generated by long time contributor Ye Yang. He took the ORF of my MRI and sub-cloned it into his vectors. Thank you! These variants will be easier to purify and give a higher soluble yield.
3) His-Taq – For specific assays Taq polymerase is preferable to something like pfu-sso7d. Generally it’s pretty difficult to find a HIS-tagged Taq polymerase, so here we are, nice and simple!
Read on for rationale and further details. Academic users can get in contact to test the plasmids!
Cheeky TEV (CTEV)
Tobacco etch virus (TEV) protease is one of the most commonly used proteases in the molecular biology toolkit. It’s highly specific, efficient and has undergone many improvements over the years. I would suggest reading David Waugh’s FAQ, his group has been one of the major contributors to the enzyme over the last two decades. What mutations have been added over the years?
(Kapust & Waugh 1999) -> Fusion to maltose binding protein (MBP) + auto-cleavage of itself out of fusion protein
(Kapust et al. 2001) -> S219V
(Lucast et al. 2001) -> S219N
(Berg et al. 2006) -> T17D, N68D, and I77V
(Cabrita et al. 2007) ->L56V, S135G
(Wei et al. 2012) -> Berg + Cabrita -> T17S/L56V/N68D/I77V/S135G
(Sanchez & Ting 2019) -> I138T, S153N, and T180A
The development of MBP as a solubility enhancing partner was absolutely critical in enhancing soluble yield of TEV without the pain of refolding inclusion bodies. Waugh’s group was actually the first to demonstrate the solubility enhancing effect of tagging proteins with MBP.
S219V and S219N were both intended to knock out the auto-catalytic cleavage of TEV, however S219V became standard due to higher stability. Interesting to note, the group responsible for S219N was led by Jennifer Doudna. Yes, CRISPR Doudna!
Berg applied error-prone PCR evolution to TEV, while Cabrita took the in silico approach, both obtaining mutations that improved solubility and stability. Wei combined the mutants of the previous researchers into a quintuple mutant, which was allegedly superior. A follow up article showed that there may be more nuance to the story, however the quintuple mutant is still a nice TEV variant.
Sanchez & Ting also took a directed evolution approach, using a yeast based platform. Their variant uTEV3 actually showed 3 fold increased activity over wild type!
So, what does CTEV bring to the table? Everything! CTEV combines the solubility enhancing properties of MBP, the auto-catalytic inhibition of S219V, the quintuple mutant of Berg, Cabrita and Wei AND the triple mutant of Sanchez & Ting. Is it risky to combine all these mutations in one package! Absolutely! Is it cheeky? Oh hell yeah it is! CTEV specific purification protocol will come shortly, however beta testers will want to use this method as a guide: 64_Waugh.pdf
As a side note, most of the previously mentioned TEV mutants are available on Addgene if you want to be a bit more conservative! 🙂
Edit: There’s a fair number of TEV variants that I failed to mention, so I’ll list any additional mutants here. Only those mentioned above were incorporated into CTEV.
(Blommel & Fox. 2007) -> C-terminal truncation to increase solubility, thanks to Priyadarshan for the tip.
(Renicke et al. 2013) -> Mutations that alter P1′ amino acid specificity
MBP-Sumo/TEV-MRI
Murine RNase Inhibitor (MRI) is one of those enzymes that you basically drink when you’re a RNA focused lab, so it was a prime target for synthesis. To make things a little more flavorful I chose Rat RI as the base (A rat can eat a mouse, therefore Rat RI > Mouse RI), and added a single amino acid change based on the following patent (https://patents.google.com/patent/US7560248), which describes a pair of cysteines which can be mutated to increase oxidation resistance. For a wonderful review of different RI variants, check out the following: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4219644/. At the time I thought the enzyme would be adequate for the purpose so I cloned it into a N-terminal 6X HIS vector and called it a day. I should’ve dug deeper, however, as reasonable soluble yield of this enzyme could only be achieved at 16C.
Ye Yang, friend and long time contributor to the blog, clued me into the better way of expressing RI: as a fusion to maltose binding protein (MBP). Ye took my pML-MRI plasmid that I had sent him and sub-cloned them into 6xHIS-MBP-TEV and 6xHIS-MBP-Sumo. Here is his purification protocol and initial results: Ye’s MRI purifcation. He based his clones on the following paper: https://www.sciencedirect.com/science/article/pii/S104659281100026X. Interesting how Waugh’s MBP fusion technique makes an appearance here too!
Ye provided me with both TEV cleavable and Sumo cleavable versions, although he personally prefers the Sumo. Ye’s plasmids will be replacing my original pML-MRI going forward.
His-Taq
I always thought that more accuracy = more better when it came to polymerases, so I never paid much attention to our old friend Taq. As it turns out, Taq and its derivatives are still commonly used in applications like qPCR, where speed is preferable to accuracy and proofreading ability.
Interestingly, all the available Taq plasmids on addgene aren’t His tagged! The old school way of purifying Taq is heating it up to drop out the E.coli proteins, precipitating it with PEI and optionally passing it over an ion exchange resin. His-tagged Taq can be purified over nickle beads, which is approachable for beginners in protein purification.
So, here we are, His-Taq available upon request!
Update on DpnI
DpnI is an enzyme that we use a surprising amount of for cloning, and I was pretty eager to get it synthesized for purification. I should have read the fine print a bit more carefully, you need a VERY specific BL21 strain for expression, one that does not methylate its own DNA. D’oh! Until I can find a way to modify the plasmid to allow expression in regular cells, I will not be including DpnI in the main plasmid pack. I will send it on request however.
It’s surprising to me, but Allergan did some optimization of TEV mutants expressed in Coli and found that more mutations was not necessarily better (see Table 3/4). They found that the T17S/N68D/I77V/S135G mutant with S219N gave the highest amount of purified protein (ca. 35 mg/L under their conditions) and was arguably the most active variant tested. Strangely, they claim that the S219N mutation was more stable than the S219V mutation, in contrast to what Waugh reported.
https://patents.google.com/patent/US8546108B2/en
Thank you for the information and the patent!
Intuitively it makes sense, stacking mutations on top of one another will eventually start disrupting the structure and function of the protein. Especially if you’re just applying them by hand, and not through some guided evolution…
Shortly after releasing CheekyTEV a Swedish dude got in touch to say he made essentially the same clone I did, completely independently! Some sort of convergent evolution through the hive mind. Anyway, he found that the “greedy” mutant was functional, so I can at least say CheekyTEV is the same. How it stacks up against the top TEV mutants? We will see! I’d love to be able to do this sort of thing full time…
I think the space of designer proteases will grow, TEV is just one of thousands of potyviruses!
We were trying to purify the P50-T4 ligase instead of our old stock T4 ligase. The yield is good, but the bands are 70kDa and 50kDa. None of these are the P50-T4 at 100kDa. After T7term sequencing, there is a mutation in frame TAC -> TAA……. ~1750 bp from N-term. That is a bad luck. It might happen when i transfer the plasmid to DH5a. for glycerol stock.
Thanks for letting me know, I will sequence the clone on my end ASAP! I don’t want to send anyone down the wrong path.
We faced the same problem.
Hi,
anyone tried His-Taq expression ?
Yup, there have been one or two individuals who have expressed it and performed PCR with it.
can you any one share the protocol of taq expression, we are getting very less yield.
We have our own His-Taq construct in pET28.
Day.1. Transform into Rosetta 2(DE3), onto ChlR and KanR LB plate, 37C overnight.
Day.2. Inoculate colony into 15 ml LB with ChlR and KanR. Don’t use old plate, like stored at 4C after transformation.
Day.3. keep Glycerol stock, which could be use directly for inoculation next time, store at -80C. Transfer 15 mL into 3L LB (not need antibiotics and we always add ~5 drops antifoam into 1.5 L LB), growth at 30C for 6-8 hours. Add IPTG at 0.25 mM and induce at 18C for 20 hours.
Day. 4. Harvest cell pellet. Resuspend by 80 mL Tris-HCl pH 8.0 40 mM, NaCl 400 mM, 10% glycerol and 15mM Imidazole. Break cells by Microfluid (Sonication is also fine). Incubate at 75C for 40 mins, mix every 10mins. Chill on ice for 20 mins. Centrifuge at 15k rpm for 25 mins at 4C. Apply supernatant to 5 ml Ni-NTA column. 30CV-40CV wash by suspension buffer. Elute by additional 500 mM imidazole. ~ 14 ml eluted sample are collected ~ 30-50 mg. yield.
Optional: Dilute 14 ml eluted sample by miliQ water. Apply to 5 ml Heparin column (~ 10 mg / ml capacity). Wash with 25 mM HEPES pH8.5 (or Tris-HCl pH 7.5), 100 mM KCl, 10-20 CV. Elute by 25 mM HEPES pH8.5(or Tris-HCl pH 7.5), 450 mM KCl (you can do 350 mM KCl, the peak will be slightly wider).
Dialysis the eluent (either from Ni-NTA or Heparin) to 10-20 volume, 30mM Tris-HCl pH 8.8, 100mM KCl, 0.1mM EDTA, 0.2% TWEEN-20, 1mM DTT, 50% glycerol.
Day.5. Collect, and store at -20C. Determine concentration by nanodrop is okay. coefficient is 1.2. 1 mg/ml is our stock. 1 mL PCR reaction we add 1-3 ul this stock, we did another diluted stock for routine.
The Heparin column is optional, I found this from my previous lab member. He left us so neat and detailed note book.
Thank you for the protocol and insight Ye 🙂 the growth and induction at less than 37C is a good technique for increasing yields, I never induce at 37C anymore.
For more reading, you can take a look at the Barrick Lab protocol for pfu-sso7d (https://barricklab.org/twiki/bin/view/Lab/ProtocolsReagentsPfuSso7d). I followed it when I purified His-Taq from a blue-light inducible vector and you get loads. I would take Ye’s advice and use Tris instead of phosphate for the buffering, if you get phosphate in your final enzyme prep it can inhibit your PCR reaction.
Hey Ye and Alex
Thats crazy yields for Taq. We totally scrap for (so little ) taq specially that we are home-brew enzyme addicts !! also how come you guys don’t add antibiotics? whats up with that ? !! we suffer so much from degradation !!
any chance we could contact you directly Ye?
I should probably set up some way for people to contact each other without posting their emails, in the meantime I can connect you two if everyone is on board.
That. Would be great Alex
Tnx a lot
The overnight culture has antibiotics and have already selected the strain. For the amplification step, i would prefer let the cell spend more energy on growing rather than struggling with antibiotics. I think you can also add 0.1% glycerol, 0.1% glucose and 1mM MgSO4 to the LB to make the cell more happy, I learned this from here, Alex’s blog.
The degradation also happened to us. I think we fix it by not using old plate as I mentioned. Fresh Rosetta transformation (or the glycerol stock) does help. Maybe before making your competent cell, the colony is also better from fresh plate or glycerol stock directly. Of course we didn’t do it side by side. Maybe just picked the right colony at the second time.
Hi Ye,
Can you please send me your taq contruct please.
Cool 😎 tnx a lot
Also i think our plasmid is not pET and thats a difference
Is your plasmid just a His-Taq in a pET?
My His-Taq is in a pet-28a vector.
Alex is that the one you sent me and Sarah or another one. No way 😎😎 our lucky day we should try it
Ya my guy, thats the one!!! Good luck with it 🙂
The one in our lab is NdeI/NotI in pET28b. Looks like a N-term His. I guess it is kind of same to Alex’s.
I was wondering if I could get a *.gb or fasta file of your taq plasmid? I would like to apply some modifications (generation of stoffel fragment and domain swapping for increased strand displacement activity) and primer design is much easier if you don’t have to guess the right sequence (and I really want to avoid primer walking…)
Email sent 🙂
Wow that was fast, thanks a lot!
I purified some DpnI in JM110, which is dam-, dcm- stain. Free from CGSC for low funding lab. The DpnI is sub-cloned into plasmid with tac promotor, so no DE3 needed. The yield is so so. It prone to degradation, looks like. But more than enough for lab use. Tested by similar protocol of Quickchange. 1 liter cell culture got 2 mL final product with comparable activity to FastDigest DpnI.
Thank you for send me the original plasmid.
Greetings Ye,
I hope you are reading the blog, I have two questions if you are available…
1- Can you please share with me (cihan.aydin at gmail) the sequences for your plasmids SUMO-MRI, MBP-MRI and Ulp1 with me if you can?
2- Have you tried to use the MRI without getting rid of the SUMO tag?
Take care,
ML
No problem man, you’re the first of all testers to manage to purify the plasmid 🙂 I’m glad JM110 worked, it’s easier to find than the one mentioned in the original DpnI purification paper.
Hi Pipette Jockey,
I just discovered your webpage today and I very much appreciate all the tricks and shortcuts and
self-brewed enzymes. I would like to try DpnI expression as well as we use a lot of it and if you
know by chance BsaI, its another one we use very regulary…
I read that you are not sending out plasmids anymore what I fully understand but maybe you can
share the sequence or link that I can get it simply synthesized myself – that would be a great help already.
Greetings dear fellow tinkerers,
I was wondering if any of you would have the sequence for the MashUP-RT. I currently obtained a pCold-TF plasmid to which I would like to subclone MashUP to check for solubility and function with the trigger factor associated with it. I would be really glad if any of you can share with me the sequence.
Take care,
ML
Hi MadLab,
I was wondering if you could share the MashUP-RT plasmid with us?
Thanks in advance and keep up the great work!
Best,
Gabor
Greetings,
As long as you can send me a FedEx or equivalent customer ID I can send it to you absorbed to filter paper – however my schedule is quite erratic so you may need to remind me a couple times 🙂
Please e-mail me at cihan.aydin at gmail using your personal e-mail belonging to the academic institution that you work in – so that I will know that I will be sharing this construct in the spirit of them being given.
Also, I cloned MashUp (thanks Alex for the sequence!) into pColdI and pCold-TF and will try purification on them this week to see if TF enhances stability and yield of the MashUp. I can also send these as soon as I verify superior expression.
Additionally, I used chaperone plasmids from Addgene (83922 or 83923) and observed a significant increase in soluble expression when induced at 18C. So you may also opt to procure them from Addgene.
Take care,
ML
Greetings everyone,
For those of you who were requesting plasmids and haven’t sent a message for the last 2 months… I was actually really close to dying and it took me like 7 weeks to recuperate but I am slowly returning to work and lab. I will probably send you plasmids somewhere mid-april provided that you can send me a DHL shipping label (because it is closer to where I live).
Take care and sorry for the extended delay 🙂
ML
hi there,
I am new to this group , so pls help me out here.
i would like to have the His Taq vector system. so what is the procedure to get it
best
Rajiv