Mashup RT Beta Update #1

“I’ve done this experiment in LB with addition (after autoclaving) of 1mM of MgSO4, 100uM of MnSO4, 0,1% glucose, 0,1% glycerol. After the culture had reached an of OD 0.9-1 , I induced the culture with 0.5mM IPTG at 20C for 25 hours. For lysis of the induced cells I used a protocol from this paper (https://link.springer.com/article/10.1007%2Fs10529-009-9977-5). 

I achieved protein yield of about 30mg of protein per 1L of culture. The yield might be higher since there is plenty of insoluble protein.

In my hands Mashup RT has the tendency to precipitate during Amicon concentration and buffer exchange.

I dialyzed to 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) is the best option, at higher protein concentration Mashup RT has the tendency to precipitate if you have 100 mM NaCl in the storage buffer.

Purified protein have activity of about 200 U/ul. This batch has no detectable RNAse activity after Ni-NTA purification.

I’ve checked protein activity by comparing qPCR Ct values of cDNA made from same RNA using RevertAID RT, SSIII and MashupRT. RT reactions were done in original buffers, reaction in revert aid buffer was done at 42C, in SSIII buffer at 52C, with oligo (dT) primer for 1h. To compare RNAse H activity of SSIII and MashupRT I compared samples treated with RNAse H to not treated.

I’ve prepared new batch of Mashup, this time I’ve made nice gel photo. I must say that this one is even higher purity then previous batch.

“Preparation and activity testing of Mash up RT   25.04.2019

We have expressed both Mash-up RT and Mash-up-cold-RT using Rosetta II cells grown at 22 degrees.  The cultures were grown at 37 deg. to an OD600 of 0.6 and then induced by IPTG and switched to 22 degrees.  Protein was expressed at 22 degrees for a further 10 hours.

Cell pellet was resuspended in lysis/binding buffer of 50 mM Sodium Phosphate, pH 8.0, 500mM NaCl, 10mM Imidazole, 5 % Glycerol.  Protease inhibitor cocktail was added.  Cells were lysed using a French press.

After a high speed the soluble protein was purified from the supernatant using Ni-NTA resin.   In brief, the supernatant was incubated with Ni-NTA resin to bind His-tagged proteins.  The unbound fraction was then removed, the resin washed and the bound protein eluted stepwise with 100mM to 500mM imidazole.

After lysis most of the protein was soluble.  Binding of the protein to the Ni-NTA under our conditions was not completed.  After extensive washing the bound proteins were eluted with sufficient purification for the first step of purification.

The eluted protein fractions were pooled, dialysed into enzyme storage buffer, filtered and concentrated. 

Some of the protein was lost during the concentration steps. It seems the protein either precipitates and/or sticks to the membranes in the centricons.

The Mash-up RT in storage after 1 step purification

We recovered approximately 9mg per L of culture

The protein shown in the above gel was tested for activity.

Test reverse transcription was carried out at different concentrations of the Mash-up RT and compared to SSIV RT.

PCR amplification of the resulting cDNA showed that the purified Mash-up RT is at least equivalently active to the SSIV.

As a second purification step to further clean up the protein the following were tested;.

Heparin column purification did not work under the first conditions we tested.  Cation exchange chromatography at pH 7.6 did result in a cleaner protein which we are further testing for RT activity and RNase contamination.”

Link to Ryan’s LinkedIn: , https://www.linkedin.com/in/yhnai/

“For the purification of MutD9 I used the chaperone plasmid pG-TF2(groES-groEL-tig). Interestingly the pG-JKE8 didn’t work for me as I think it may have something to do with adding the appropriate amount of L-Arabinose. Having said that this pG-TF2 plasmid still requires growing the culture to OD 0.6+ before induction with Tet and IPTG.

Eventually, I switched to chaperone plasmids from Addgene – pBB540 and pBB541. They work well with pET plasmid (carrying MutD9 sequence) and lac repressor Autoinduction system using the popular ZYM-5052 (lactose 0.2%) media.

I used the Ni-NTA system (with Tris Buffered Saline 250mM NaCl pH 8, 10 mM Imidazole, elute at 0.5M Imidazole) with a gravity flow column. Buffer exchange was performed with Amicon MWCO spin column.”

Feedback 1:
“We obtained a reasonable yield of Mashup-RT (0.2 mg / 50 ml culture) using BL21 cells induced with 0.8 mM IPTG at 16C for 24 hours. Crude lysate was purified one-step NiNTA.

We found that the prep was inhibited by the buffer from the ABI kit, but the prep works well in universal buffer (75 mM KCl, 50 mM Tris-HCl 8.3, 10 mM DTT, 3 mM MgCl2). All first strand reactions were done at 37C for 2 hours, with similar results being observed at 50C.

We also ran qRT-PCR reactions with two housekeeping gene primers and two single copy gene primers. For the housekeeping genes, the Cq from homemade buffer/MachUp is ~ 2 larger than from the ABI kit(like 20 vs 18). For the single copy gene, the homemade one always got lucky—- smaller Cq (like 35 vs. 37).”

Feedback 2!:
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.

“A couple of years ago, I synthesized reverse trancriptase with mutations gathered from ssII, agilent and fermentas patents, which I purified and used successfully in my work. I advise you not to use autoinduction media during MMLV expression. In my experience, 70-90% of the MMLV-RT ends up in the inclusion bodies, and induction using lactose (galactose) promotes their formation. I conduct induction in standard BL21 cells without the 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 add IPTG to a final concentration of 0.2 mM. Induction takes 5 hours. On average, from 1 liter of culture, 5 mg of purified MMLV is obtained (Ni-IDA plus desalting) with an activity of about 200,000 U/mg.”

“I earlier tried purifying Mashup and the Pfu-sso7d side-by-side using the Pierce Pulldown PolyHis Protein-Protein interaction kit (21277) for small scale preps. We had the kits on-hand for purifying ProteinA-ProteinG-MNAse for Cut&Run. Anyways, I got workable Pfu but no Mashup came down at all. I grew Pfu just in LB, but grew the Mashup in LB+. I noticed that the LB+ / Mashup cultures seemed to continue dividing or at least the culture looked a bit cloudier (the CRB was sterile filtered and everything done around a flame, but something could have gotten in there, I suppose).

So today I retried Pfu and Mashup, testing with and without CRB (i.e. LB vs LB+) and lysis using the lysis buffer in the kit versus sonicating in the buffer you recommend. For growth, all 4 Pfu cultures looked much less dense/cloudy after induction than the 4 Mashup cultures, irrespective of whether there was CRB. Further, the 2 Mashup cultures treated with lysis buffer had really puffy, gooey pellets, and the washes on the column got very foamy. I don’t think there is DNAse in the lysis buffer, so it could be that there was a lot of DNA or RNA making it behave strangely, but that this was sheared up in the sonicated samples. All four Pfu preps seemed ‘normal’.

I ran out samples on TGX stain-free gels and attached a figure. I recovered more Pfu with sonication, though it seems to prefer induction without CRB. Mashup prefers CRB but only seems to bind the column with sonication. Absolutely nothing in the elution using lysis buffer (I guess it could be stuck on the column…).