Published: July 15th, 2019 Last Modified: April 16th, 2020
It’s awesome how far we’ve come as a little community in less than a year, from the first inception of Mashup to the first feedback from the beta testers. It’s finally time that I present our take on it’s purification as well as characterization of it’s activity and improvements to the RT buffer.
Considering that Mashup is now fully integrated into our lab’s workflow, I can comfortably call this project a success. Grab a coffee, get settled in, let’s get to all the juicy details!
As always, credit where credit is due. The bulk of the purification, optimization and characterization of Mashup-RT was performed by an undergraduate thesis student, Jayshna Malloo. Thank you so much Jayshna, you’ve set a high standard for what can be accomplished in just a few short months! We wish you all the best in the future.
Purification
!!!UPDATE!!! If you don’t want to hear me ramble, consult the abridged, most up to date protocol: PDF, DOCX
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Buffer ListStock Solutions
1M Tris-HCl pH 8.0
1M Tris-HCl pH 7.5
10% Triton X-100
1M Imidizole
0.5M EDTA pH 8.0
Catabolite Repression Buffer (CRB)
25% Glycerol
25% Glucose
1 mM MgSO4
0.1 mM MnSO4
Filter sterilize, aliqout, freeze, Use 4 mL / L of media
2.5X Lysis Buffer
0.75M NaCl
62.5 mM Tris-HCl pH 8.0
25% Glycerol
1.25% Triton X-100
4X Storage Buffer (No Glycerol)
600 mM NaCl
200 mM Tris-HCl pH 7.5
4 mM EDTA
0.4% IGEPAL CA-630
Starting off with the choice of strain, we had two options based on feedback we received. We tested both BL21 DE3s and BL21 DE3s with pG-TF2 chaperone plasmid. While pG-TF2 does certainly increase soluble amounts of protein, it isn’t strictly necessary, sort of a smoke em’ if you got em’ type situation. Our results echoed what previous testers found, that high concentrations of Mashup actually cause precipitation and protein loss, so there’s a good balance somewhere. One 200 mL purification already gets you thousands of reactions worth of enzyme, so no need to go crazy.
Cells were transformed with the Mashup plasmid (Plated on LB/Kan at 50 ug/mL final), and inoculated into a small 3 mL LB culture to grow overnight overnight at 37C.
1 mL of this culture was used to inoculate 200 mLs of LB+ in the morning. LB+ is prepared by autoclaving regular LB then adding 4 mL filter sterilized CRB.
The large culture was incubated at 37C in a shaker at 275 RPM. OD was monitored until it reached 0.8-1.0 and was induced with 0.5 mM IPTG (pG-TF2 chaperone requires pre-induction at 37C for 20 minutes with 5 ng/uL final conc. of tetracycline). Induction occurred at 28C for 20 hours. Ideally you would incubate at 22C or even lower, you get more protein that way, but 28C still produced plenty of protein.
*** Some lysis buffers may be incompatible with Mashup, see Colin’s feedback in previous post ***
Cells were harvested by centrifugation, washed once with 1X Lysis Buffer (20 mM Imidizole) then resuspended in 5 mL of 1X Lysis Buffer (20 mM Imidizole) 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. Lysate was centrifuged at 20,000 xg for 30 minutes at 4C to pellet cell debris. Clarified lysate was then loaded onto 0.5 mL of Ni Sepharose Fast Flow beads (use whatever you got) that had been equilibriated with 1X Lysis Buffer (20 mM Imidizole).
Column was washed with 5 mL of 1X Lysis Buffer (50 mM Imidizole) , then eluted with 5 mL of 1X Lysis Buffer (500 mM Imidizole). 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.
The eluted protein was brought up to 10 mL with 1X Storage Buffer (0% Glycerol) and loaded onto a 50 kDa Sartorius concentrator column. Sample was centrifuged at 3200xg and 4C until the level had decreased to 2 mL. The protein was brought up to 10 mL again with 1X Storage Buffer (0% Glycerol) and this process was repeated for a total of 4 times. Remember to mix your sample VERY, VERY well with a 1 mL pipette between concentrations. Also, It’s a good idea to take samples before/during buffer exchange, over-concentrating can cause you to lose your protein and you want to know when/if it’s happening.
After the fourth buffer exchange, the protein is brought up to 10 mL with 1X Storage Buffer (60% glycerol) and concentrated down to 2-5 mL (If your liquid level is going down, keep going, if it’s stable then proceed). Then, the protein was brought up to 10 mL with storage buffer (50 % glycerol) and concentrated until the liquid level stopped dropping. Mix very well and quantify with BCA assay. After quantification with BCA is complete, add DTT to 1 mM final concentration, aliqout into 500 uL portions and store in the -20C. This is your undiluted stock, so take care of it!
Whew! What does our protein look like after IMAC purification and concentration?
Wonderful! Be cautious of over concentrating here, you could lose it! Not bad after just a nickle column and 50 kDa column, eh?
Quality Control / Measuring Activity
So, after purification we checked out whether our RT had any RNase activity. Total RNA was incubated with various dilutions of the purified Mashup in 1X First Strand Buffer at 37C for 3 HOURS!
Excellent, little/no RNase activity at even undiluted concentrations.
At what dilution is the Mashup active? RT for a ~1 Kb plant mRNA at 50C with 500 ng total RNA and First Strand Buffer.
How sensitive is Mashup RT to low amounts of RNA? RT at 50C with First Strand Buffer.
Thermostability was also tested with qRT-PCR Ct value as the metric.
Buffer Optimization
Considering that 5X First Strand Buffer has been used for decades, and that Superscript 4 has it’s own proprietary buffer, we reckoned that we could push the performance of Mashup-RT with buffer optimization, take a look at the raw results.
Sorry for poor quality, these are the updated optimization results. T = Tris M = MOPS H = HEPES. Interesting that MOPS/Tris outperforms either on its own! Here’s our best buffer to date:
5X Mashup Buffer (5X MB)
125 mM Tris-HCl pH 8.3
125 mM MOPS pH 7.9
300 mM KCl
20 mM MgCl2
25% Glycerol
0.03% NP-40
How does it perform vs the tried and true 5X First Strand Buffer, and Superscript 4’s proprietary buffer?
So, there you have it! A facile purification protocol and optimized buffer! I’ll be adding and polishing this page as more results come in. Comparisons to Superscript 4 are being performed, but Mashup can easily hold its own.
I will be attempting FPLC based purification but looks like gravity flow is good enough to produce buckets of RT! A small 200 mL purification produced 60,000$ worth of RT, considering 1 uL of Superscript 4 is $12 USD.
do you know simple method for hot star taq polymerase preparation?
Check out Biotechnol Lett (2006) 28:1939–1944
DOI 10.1007/s10529-006-9178-4
Which gene you used for validation , can you share sequence of primers
If you’re validating the enzyme I would use whatever primers you have on hand that produce a product of a decent length (~1kb). Also the RNA should be at a decent abundance in the total RNA of your organism of choice.
If you’re still curious we used the ORF of pokeweed antiviral protein (PAP) and pokeweed total RNA for validation, but for the vast majority of people that’s not really relevant or readily available.
Thanks for all this amazing work!
So from you experience and many others, it seems the protein is quite sensible to the concentration process (or simply being concentrated). Would you say to exchange buffer, dialysis could be a solution to avoid the multiple concentration steps you used to change the buffer?
Also I’m curious, what kind of yield did you get for these 200 ml of culture ?
Thanks
I’m glad you enjoy it 🙂
I would say that almost any other method of buffer exchange would be more efficient and cheaper, I’ve been playing around with G25 desalting columns myself as a form of buffer exchange. Also it’s not unlikely that the sharp changes in concentration are what sometimes trigger mine, and others, precipitation.
I’m barely learning to walk in the protein field, so I have a pretty brutish approach to the purification, lots of room for finesse and efficiency.
The yield was approx 1.5mg, recently I got 7mg from 1L. Not mind blowing, but still, quarter of a million reactions. An expert would likely be able to push this further.
Thanks for the very quick reply!
I’m going to give it a try using your protocol in the coming days/weeks
Great work. I noticed a lot of commercial RT reaction require additional DTT (5mM in general). Is that important?
Are you referring to the storage buffer? When I was trying to determine the best value I saw commercial offerings between 1 and 5mM, I figured why not try to get away with less? So far I haven’t noticed loss of activity in my last batch, and that’s 1.5 years old. I always make a fresh batch of DTT for every purification. But yeah, I think you can bump up that number without too much trouble, spice it up a bit. With that said I haven’t tried it, I bet there are a few ways to squeeze a bit more performance out of the enzyme.
Waah, flash reply. I meant the reaction buffer. Like, superscript iii, it comes with a 0.1 M DTT and suggests add as 20x in final reaction. Also I found this patent from Qiagen US20040110201A1. Also, 5 mM DTT added and 20mM LiCl .
Yes, the reaction protocol for Mashup is the same as SS3/SS4, where you add 1uL of 0.1M DTT to a 20uL reaction volume. I’ve included a typical protocol in the abridged purification protocol, but it is the standard RT reaction we all know and love! 🙂
Here is my real question. Why add DTT separately? Actually, I forgot to add DTT with our Superscript iii reaction and didn’t notice any difference. I am wondering why people not included it in the 5x buffer or ignore it. Then I found here 🙂
It’s one of those things that your granddaddy or grandmammy did back in the day, and nobody bothered to really question it. Here we are, decades later, wondering why add DTT?
Some people swear by adding the dNTPs before the heat denaturation,some add the dNTPs after…both ways work…
Same thing with the RNase inhibitor, you can get into a fistfight with people over whether to include it or not, people do RT without inhibitor all the time.
Now that I think about it, the DTT could have been kept in the recipe as a RNase inhibitor, it’s pretty effective at that but it’s a lesser known ability. Before widely available milliQ water was a thing you probably had a harder time reducing RNase activity. People who kept adding DTT were more successful, passed along the protocol…
Something to be said about scientist psychology, for sure.
Hi Alex,
Any idea what’s the pI of Mashup RT?
Theoretical pI/Mw: 9.44 / 76.4 kDa
Thanks. I was getting some RNase activity and I will be trying GEC to further purify it. The RNase activity could be due to the 30 kDa cut off coulmns, as I was unable to get 50 kDa cut offs.
Thanks so much for all of this info and reporting on the Mash-UP RT purification/activity test etc.
I have a question, we are having an issue with overestimation of the protein concentration via A280 absorbance. What method are you using to estimate protein concentration of the sample?
Thanks!
Kita
We use the BCA assay for quantifying the protein, Bradford doesn’t play nice with the RT. You could also run a BSA gradient on a gel to get another estimate 🙂
Sounds good thanks!
Also, I used a desalting column as a secondary purification to get rid of smaller proteins and to buffer exchange the sample. Works well and this is a fast way to buffer exchange. At this point I don’t know exactly how much I lost on the column (probably around 20-40% based on gel) – but the outcome is relatively pure and less finicky then doing the buffer exchange on Amicon/Vivas concentrator (I also did this – but filter got clogged pretty quickly).
Hello!
I have messaged you before to test out some ideas about purifying enzymes, and recently I thought about Mashup-RT purification, and maybe its tendency to precipitate is not a bad thing?
Have you tried precipitating it with 80% cold acetone to get an acetone powder and then resuspend it at a necessary concentration?
I got inspired by the fact that apparently Taq polymerase survives being acetone-d.
doi: 10.15414/jmbfs.2018.7.5.445-448
That’s just an idea, but I am ready to test it out if you are okay with sending plasmids for me, or you can test it yourself if you are not too busy!
Thanks for the wonderful post. We have purified our RT recently and are in the process of characterizing and optimizing it. I am curious how units of the RT enzyme are determined. Commercial suppliers mention a radioactivity based assay for unit assessment. Can you suggest other methods?