NEB TV Ep. 30 – Cell-free Protein Synthesis

Cell-free protein expression enables the synthesis of a wide variety of proteins in just a few hours, and can be used in a variety of applications, including protein engineering, expression of toxic proteins and diagnostic assays.

Script

Deana Martin:
Welcome to NEB TV. Today we are talking about cell-free protein expression. In our Science in 60, Colby Stoddard from NEB's Business Development Team gives an overview of the benefits and applications of cell-free expression. Then we will hear from Zach Sun from Tierra Biosciences and Keith Pardee from the University of Toronto, and they will discuss some interesting applications that use cell-free expression in their labs. And lastly, we will hear from Paula Magnelli here at NEB, and she will discuss the workflow and benefits of NEB's new NEBExpress cell-free E. coli expression system. Let's get started.

Colby Stoddard:
Living cells can be genetically modified to perform novel functions such as the production of recombinant proteins. However, these genetic modifications often conflict with normal cellular processes and result in sick cells or mutational events that negatively impact research efforts. These shortcomings can be overcome through removing the cellular membrane, which leaves a lysate that is rich in enzymes that can perform both transcription as well as translation. This also removes the competing interests between the cell and the researcher and creates new opportunities for the production of toxic byproducts, point-of-care diagnostics, as well as synthetic biology applications.

Colby Stoddard:
The use of cell-free lysates also simplifies the research workflow since one can directly introduce DNA without the need to perform transformations and clone propagation. Lysates are also readily manipulated in high throughput automated workflows and allow for researchers to explore more biological designs leading to new deeper insights into biology.

Zachary Sun:
Cell-free technologies are a pretty old technology, they've been around earlier than we can engineer cells, but there's really three applications that I see cell-free has been currently useful far. One is to make things. So there are commercial companies that do this, a lot of R&D labs that do this, but if you have proteins that are toxic or otherwise would be hard to make in cells, cell-free is a really good way to not only make the product that you could then use downstream, but also understand how to make the product. So making stuff, I think, has been a historical use, but there are also two other areas where cell-free is quite useful.

Zachary Sun:
One is in understanding stuff. What I mean by this is if you have a set of circuit or a protein or even a cluster that makes a small molecule, understanding how that system works, that genetic system, cell-free is a great way to do that. And that's kind of what's sparking from a cell-free biology – really to start to decipher some of the rules of biology and rules of these systems.

Zachary Sun:
And then finally, in sensing stuff. Cell-free as an application itself, if you want do biological computation, say to be able to sense something in an environment, do a biological response, cells are really good at that, but it's very hard to program that into kind of keeping cells alive for in-field application. But cell-free as a sensing material, is quite powerful too. It allows you to do computation without having to maintain a cell. And so I think these three applications are pretty powerful.

Zachary Sun:
Now, many of the challenges I think currently are around the applications set of cell-free, right? It's really kind of education. Say, if you're doing a set of work in cells, how can cell-free help you speed up that process, and how do you understand the results that you collect in cell-free, how it might influence what you do in cells, or conversely, maybe a result isn't applicable to cells. And so, I think education is a big part of this, kind of understanding when cell-free can come in handy and come into use.

Zachary Sun:
Tierra Biosciences is a company based out of Berkeley. We're using cell-free to effectively do functional genomics. When we think about cell-free, we think of it as a really good platform for understanding how to make things, and then making things to test. And so, if you have a bunch of DNA and you need to be able to make the protein that's not working in your cell, or you have a lot of proteins you want to make, for us, we've built out a system that's driven by cell-free synthetic biology, some AI and automation that's able to really quickly take digital sequences of DNA and then say, "What does this make, and what does it do?" And understand really the process it goes through transcription translation and such. So the applications for this are really in functional genomics and also kind of in understanding complex systems.

Keith Pardee:
My name is Keith Pardee. I'm an assistant professor in the faculty of pharmacy at the University of Toronto and the Canada Research Chair in Synthetic Biology and Human Health.

Keith Pardee:
So the main challenge with mosquito-borne illnesses is really detection, right? If you have early detection that there's virus in the mosquitoes that is soon going to translate into infections in people, and the sooner you can identify the infection in people, as well as in mosquitoes, the more quickly you can bring those outbreaks under control and reduce the spread of the infection.

Keith Pardee:
And so, providing tools for diagnostics that are more portable and low cost, our goal is to help in that effort. The largest amount of product we use from NEB is PURExpress, which is their recombinant cell-free transcription and translation system for protein expression. And we use the system to run our gene circuit-based diagnostics, and also for running the portable manufacturing of drugs.

Keith Pardee:
And what we do is we freeze-dry PURExpress and embed it into paper or make pellets out of it, so that we can run these synthetic biology applications outside of the lab. And the goal here is to decentralize medical or healthcare capacities that are usually only in hospital settings, out into the real world as diagnostics and the manufacturing of drugs.

Keith Pardee:
And one of the largest efforts we do is in diagnostics, molecular diagnostics, where we use the genome of a pathogen as a barcode for identifying the presence of that pathogen. And one of those projects that's sort of our flagship project is for the detection of Zika and Chikungunya in Latin America. And so, this project involves eight labs in five countries. And our partners in Brazil, Bogota, or Colombia, and Ecuador are collecting samples and running our technology down there to determine the presence of the virus and basically establish the assay.

Deana Martin:
So we're speaking with Paula Magnelli who is an application and development scientist here at NEB. Hi Paula.

Paula Magnelli:
Hi Deana. How are you?

Deana Martin:
Good, thank you. So could you tell us a little bit about the challenges associated with extract-based cell-free expression?

Paula Magnelli:
I would say the biggest challenge is variation. When you are trying to express a protein using a cell base in vitro system, it is difficult to obtain the same result every time. Another common challenge is the fact that in a cell-free environment, some proteins have a difficult time with folding and with the formation of the correct disulfide bonds. And a final challenge is the concern that many scientists have of whether this type of reaction that is in vitro, is not a live cell, will be able to achieve the high level of proteins expressions that they require for their experiments.

Deana Martin:
So you were involved in the development of these new NEBExpress cell-free expression, correct?

Paula Magnelli:
Yes.

Deana Martin:
Could you talk a little bit about some of the advantages of using that kit?

Paula Magnelli:
Yes. One of our main goals when we developed this was to reduce the variability. This is important because in a protein evolution experiment in the discovery of a new protein variation, you need to capture those small differences in performance, small differences in expression levels, and if a system has too much variability, that will means that you don't really know if the differences that you observe are real or is just error.

Paula Magnelli:
Another feature that we really wanted this kit to have was compatibility with the disulfide bond enhancer. It assists, in combination with the NEBExpress kit, it assists proteins that have a difficult kinetics, difficult time to achieve the correct folding to form the right amount and the right combination of disulfide bond in the protein sequence. Therefore, what you obtain is not only some yield of protein, the protein that you get is the in the right form, is the active form. So that is a nice perk. It's great to have.

Deana Martin:
Great. And when you're talking about yields, how much protein are we talking?

Paula Magnelli:
A typical protein... I mean, not all protein is expressed at the same level, but I would say that the typical protein makes about a half a milligram per ml. So that will be the concentration that you get at the end.

Deana Martin:
That's great. And you can also extend incubation time, correct?

Paula Magnelli:
Yes. That is something that is a very, very nice feature. Many proteins fold better or express better at very low temperatures, but the concern with the cell-free system is whether you could extend the incubation as you do with live cells, make a 24 hour incubation at very low temperature so your protein can express and can fold correctly. And that is possible.

Paula Magnelli:
And something that is very interesting to us is that we have seen, for some targets, that in a short incubation time, of course at 25 degrees for instance, your yields are lower and that's typical. But those reactions, when they are incubated at 24 hours, the final yields can be even higher than an incubation at 37 degrees for the same protein. So that gives you a lot of flexibility. You can choose to go short at a higher temperature, or go long at the lower temperature. And depending on the goal of your experiment, the throughput or your experiment, you can have, with one kit, this flexibility. And I think that it satisfies more different types of experiments, scientific projects, which has... You don't need to think, "Oh, which one it is." It does everything. So we are actually very proud of those features.

Deana Martin:
So can you walk us through the workflow for this product?

Paula Magnelli:
Yes, sure. The workflow is very simple and that was something that we... on purpose. This is by design. The kit provides four components plus a positive control plasmid.

Paula Magnelli:
So the reaction set-up is very simple. First, the reagents that are stored at -80 degrees have to be thawed on ice. Once they are completely thawed, each reaction is assembled by mixing. The cell lysate, the extract, the protein synthesis buffer, the T7 RNA polymerase, and the RNase inhibitor. And of course, your DNA or RNA template that is for your protein of interest. And also, if the user wants, the reaction control can be performed using our positive control plasmids. And that is incubated at the temperature that you want, typically 37. You can use a different temperature for two to four hours, and then the reactions are ready for analysis.

Paula Magnelli:
And now the advantage is once that reaction is complete, the samples are ready to run on a protein gel. The components of the NEBExpress don't interfere with protein electrophoresis, so there's no need to do an acetone precipitation or TCA precipitation before running the gel.

Deana Martin:
Thank you so much for joining me today.

Paula Magnelli:
You're welcome. It was a pleasure.

Deana Martin:
Thank you so much for joining us today. If you would like to learn more about any of these products available for cell-free protein expression, you can use the link below where you can get access to brochures, feature articles, and other informative content. As always, if you have any suggestions for future episodes, please let us know.


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