So You Wanna Make Ligase, eh?
Few things bring the gentle molecular biologist to anger, but the experience of setting up your perfect ligation reaction only to find the DNA ligase tube as dry as the agarose gel that’s been on the bench for 3 weeks is one of them. You’ve put hours of work into the primer design you don’t trust your undergraduate to not mess up and several units of your highest fidelity polymerase (blessed be NEB’s Q5 hot-start) amplifying your gene inserts. You’ve even generously used half of your lab’s supply of uncommon restriction endonuclease in an effort to get your beautifully designed DNA construct, “faster”, for your PI. After all, when they were a grad student they would have had it done in less than 2 weeks! How then did you find yourself in this position: alone and without even a uL of molecular glue?
Perhaps it has to do with your lack of forsight, or maybe there’s a spare tube stashed away in your colleague’s -20C freezer box. Is it your fault? No- it must have to do with the simple fact that T4 DNA ligase, one of the most easily produced and common workhorse enzymes in molecular biology, costs an outrageous $64.00 for 20,000 units!
Figure 1. Highway robbery
Now I hear you say, “but certainly 20,000 units of T4 ligase is fair! How many units could one graduate student use?”, but I ask you to cast your eyes upon the blasphemy above once more. The concentration, 400,000 units/mL (400 U/uL), means that your $64.00 order will net you only 50 uL. God forbid you ordered the 2,000,000 units/mL (2,000 U/uL) tube, a measly 10 uL. Considering the manufacturer protocol calls for 1 uL of enzyme (at 400 U/uL) to be added per reaction and each ligation experiment typically consists of:
- 1:1 Vector:Insert Experimental
- 1:2 (or 1:3) Vector:Insert Experimental
- Backbone only –
- Insert only – (optional)
- No ligase -,
your sweet T4 ligase will not be lasting long- perhaps 5 ligation attempts.
Now, to be completely transparent we should note that diluting and using lower concentrations of ligase with overnight incubation times is a viable alternative, however overnight incubations have been noted to decrease subsequent transformation efficiency by promoting long, linear DNA strand formation. Additionally, to use this strategy you will want to tackle the whole issue of unintuitive ligase unit definitions, per NEB – “One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (5´ DNA termini concentration of 0.12 µM, 300- µg/ml) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1X T4 DNA Ligase Reaction Buffer”. I won’t pretend this didn’t take me a few takes to understand conceptually and at least 30 minutes to see as any more practical than, “Just use one uL of stock.”
Anyway, to be honest my complaints about price and concentration are just a convenient excuse to procrastinate my dissertation work by manufacturing and validating a much cheaper (and sexier) alternative: recombinant p50-T4 ligase fusion. If you’re like me and want to be set on ligase for at least the next 4 PhD generations in your lab then join me on this adventure. In the end you’ll have so much ligase that you’ll be ethically bartering with other labs for reagents, undergrad labor, and perhaps even the ever-coveted RPMI 1640, 500 mL, cherry flavored.
What is p50-T4 Ligase
To put it simply, p50 is the somewhat promiscuous DNA binding domain of the ubiquitous transcription factor NF-κB. It also serves as the dimerization domain for the mature NF-κB protein, a heterodimer of p50 and p52. The natural palindromic sequence recognized by p50 binds to it with an impressive Kd ~8 pM, but nonspecific dsDNA also has a Kd ~5.7 nM. As noted by W.M. Patrick et al., the authors who conceived of this recombinant ligase originally, fusion of p50 to T4 ligase resulted in approximately 7-fold and 1.6-fold improvements in cohesive-end and blunt-end ligation, respectively .
If you’re familiar with recombinant fusion enzymes possessing enhanced activity- recently innovated high-fidelity polymerases such as Phusion come to mind- then you may find it initially puzzling that the holy grail of non-sequence specific DNA binding proteins sso7d was not utilized over p50. Saccharolobus solfataricus sso7d is remarkable in its activity and stability, but in that stability lies its weakness for this application. For efficient transformation of a ligation mix the ligase must be heat-inactivated and its grubby grip on the vector released. Sso7d happens to be so thermally tolerant that even incubation at 95C for 15 minutes is not sufficient to release its grasp fully, hence its utilization in PCR. p50, on the other hand, denatures at a (relatively) cool 65C in only 10 minutes which is conveniently the same conditions that denature T4 DNA ligase.
A number of other DNA-binding domains were fused to both N and C-termini of T4 ligase by the authors, but overall p50 showed robust and consistent activity and so this is the version I decided to manufacture for my lab. I highly encourage you to read the original paper, though .
The Goods (Protocol)
Note: this protocol is for overexpression and purification of p50-T4 ligase in E. coli from pET28-p50.T4ligase. The ligase has an N-terminal 6xHis tag. It’s pretty robust, yielding around 10-20 mg/L of culture in my hands but I haven’t tried it with other purification schemes so results may vary.
Transformation and Culturing (Days 1-3)
- Transform BL21(DE3) E. coli or equivalent with pET28-p50-ligase vector.
- (Optional) Transform DH5alpha E. coli for a glycerol stock for future generations.
- The following day screen colonies and, on confirmation of plasmid identity, inoculate 6 mL LB supplemented with kanamycin. Grow overnight at 37C.
- The next morning, inoculate 2x2L shaker flasks containing 500 mL LB + kan with 3mL of starter culture.
- Grow at 37C with shaking to an OD600 of ~0.7 (about 2.5 hours).
- Induce with IPTG to a final concentration of 0.4 mM (2 mL/flask of 100mM stock).
- Incubate at 26C overnight (I had success sleeping in and going for 18 hours) for protein overexpression.
IMAC Purification (Day 4)
- Collect your cultures in centrifuge bottles and centrifuge 5251xg/15 min/4C.
- Resuspend each pellet in 25 mL of IMAC lysis buffer (see above) and split 1 tablet of Roche cOmplete Protease Inhibitor Cocktail tablet between the two fractions.
- Sonicate each fraction as follows: 10s on, 30s off, 30% amplitude, for 15 cycles. Verify bacterial lysis on microscope if you find sonication to be arbitrary black magic as I do. Note: Remember to keep your culture in a large ice water bath to prevent overheating of the sample and denaturation of your ligase.
- Pellet insoluble junk by spinning at 10,000xg/30 min/4C. You might have to aliquot your suspension into 1.5 mL Eppendorf tubes but trust me, if you try a lower speed for even an hour to avoid the tedious work you will reap only cosmic punishment during the subsequent filtering step.
- Syringe or SteriFlip filter your supernatant through a 0.2 um membrane. Alternatively, if you did not take my advice, wait an hour for your sample to pass through before finally giving up and returning to step 4 having wasted 2 hours.
- Add a 1 mL bed volume of Ni-NTA resin to a 50 mL disposable column. As the resin is a 50:50 slurry of resin to ethanol, make sure you are adding 2 mL slurry.
- Equilibrate the resin with 5x bed volumes (5 mL) of ddH2O, followed by 5x bed volumes of lysis buffer.
- Incubate the filtered supernatant with the equilibrated resin on a rocker for 30-60 minutes.
- Wash with 1x bed volume lysis buffer until the A280 of the flow-through levels off.
- Wash again with 5x bed volumes of wash buffer until, again, the A280 of the flow-through levels off.
- Elute 5+ fractions of 1x bed volumes elution buffer each, incubating the column for 10 minutes before each. Verify presence of p50-T4 ligase (~95kDa) in all fractions via SDS-PAGE. Pool eluate fractions containing bulk of yield.
- Concentrate your ligase with an Ultracel 30kDa spin concentrator column and, once sufficiently reduced in volume, add glycerol to a final concentration of 40-50% to prevent freezing in the -20C.
- Verify the concentration of your ligase with Nanodrop, a Bradford assay, or both. I concentrated my stock to around 5 mg/mL and found the ligase worked effectively at final concentrations between 0.3-3 uM, with minimal activity below 30 nM.
Activity of Purified Product
I verified the ability of p50-T4 ligase to join linear into circular dsDNA products with a very simple cohesive-end ligation test.
Using what I had on hand, the plasmid pSNAP-tag-m (5802bp) was linearized with XhoI overnight. The resulting linear plasmid was column purified and then ligated according to the standard protocol in NEB T4 ligase buffer with p50-ligase concentrations ranging from 3 uM to 3 nM, with both a no ligase negative and 400 units commercial NEB T4 ligase positive control. Reactions were in 20 uL total volume and were allowed to react for 10 minutes at room temperature before being inactivated at 65C for 10 minutes.
Note: When I tried to skimp on the deactivation by only incubating 5 minutes at 65C the resulting gel was disfigured and uninterpretable.
The ligated bands with our produced p50-T4 ligase look a bit blurry compared to the commercial ligase but I suspect this may have to do with insufficient heat-inactivation or more probably linear dimers and trimers of the plasmid (~12+ kb). I will be repeating this experiment in the near future with lower DNA concentrations as well as: 1. overnight incubation vs 10 minute incubation, 2. a heat-inactivation series at 65, 70, 75, 80, 85, 90, and 95C, and 3. a directional insert with 2 different sticky ends and subsequent transformation into E. coli.
Update on Activity After Storage & Transformation of Ligated Products – 2/5/2020
After working with this recombinant ligase for the last few months I am happy to report that our lab has switched over completely from commercial ligase (sorry NEB)!
Additionally, I successfully transformed numerous ligation products with no notable drop in efficiency.
At this point I don’t have much more to add regarding this project. It was fun and if you want a copy of the plasmid for academic use send me a message and I’ll be happy to mail you over a copy, free of charge.
- Wilson, R. H., Morton, S. K., Deiderick, H. A., Gerth, M. L., Paul, H. A., Gerber, I., … Patrick, W. M. (2013). Engineered DNA ligases with improved activities in vitro. Protein Engineering, Design and Selection, 26(7), 471-478.