From Templates to Discoveries: Using the NEBExpress Cell-free E. coli Protein Synthesis System for Protein Generation & Screening
Explore the NEB Express Cell-Free E. coli Protein Synthesis System in this informative webinar. Presenters discuss the benefits of cell-free protein synthesis, including its efficiency in converting DNA to protein, and acceleration of the protein discovery process compared to traditional cell-based methods.
Script
Welcome everyone, and thank you for attending this webinar from New England BioLabs. We're excited today to present From Templates to Discoveries: Using the NEBExpress Cell-free E. coli Protein Synthesis System for Protein Generation and High-Throughput Screening. Today, we will cover what cell-free protein synthesis is and how you can use it to get from DNA to your protein of interest, what advantages cell-free systems bring to scientists, how users like you set up and control experimental conditions. We will show examples of real use applications, and after we finish, we will have a live Q&A session. And for that, at any time during this presentation, you can send your questions via the chat box.
Hello, my name is Paolo Magnelli. I will be one of the presenters today. I am a principal development scientist, and I've been here at NEB for over 17 years, developing new reagents for proteomics, glycomics, and also for cell-free protein expression technologies. And just so you know, I am also one of the scientific experts that you talk to when you call or write on our tech support system. It's my pleasure to have you today here. Welcome.
Emily Chen:
My name is Emily Chen, your other presenter today. I have worked at NEB for two and a half years as a production scientist and can also be reached as a scientific expert for technical support. In this role, I support the manufacturing of the different protein expression and purification products, which are made and tested at our facility. This includes our cell-free protein synthesis kits, one of which you'll hear about today.
Paula Magnelli:
So in a modern molecular biology, you not only need to know the sequence of your gene, you also need the protein to understand its biological function. So in this scenario, time is critical. When you compare a cell-based protein expression workflow with a cell-free workflow, you can see two things. First, that cell-free workflows minimize processing time compare with say protein production in E. coli, which can take days, but also cell-free workflows simplify the protein discovery campaigns. Cell-free shortens the time from DNA to protein, and you can have purified product within one day to get the answers to your questions much faster. So we will show you next why this is the case.
I just mentioned that it takes several days to express proteins in vivo, as it's shown here in the upper part of the image. But not only that, expressing recombinant proteins uses specialized instruments and supplies. You also need expertise to handle cells and cultures for a successful experiment. First, your cells must accept the cloning recombinant DNA. This is what we call transformation. Then the cells must be able to synthesize the protein in large quantities. And sometimes this is a burden for the cells. For instance, when the protein is toxic. Also, the timing must be right. To induce our expression, you need to do it at the right growth stage. So for reproducible results, each step must be optimized for each different target. In contrast, and if you look at the lower part of this cartoon, cell-free protein synthesis is simple to use and easy to optimize because it's an open system. In the next few slides, I will show you the two approaches in a little bit more detail.
Here you see the resources needed for a traditional in vivo workflow. You need space to accommodate instrumentation and storage, equipment, capital investment, maintenance, and expertise to operate, consumables, media, plastic, water, and energy. You will have to handle high volumes of biological waste, and you will have to secure biosafety permit. But the thing is, in many cases, you do not need these dedicated setup, or you might not have the resources or the budget for this equipment and infrastructure. So how can your projects be completed in a simpler, easier, and more efficient way? The answer is, as you might have guessed, cell-free protein synthesis. Why is cell-free protein synthesis so simple and easy? Because it is just two reactions coupled together in a tube. It's an in vitro transcription reaction coupled with an in vitro translation reaction. This means that the process of mRNA synthesis and protein synthesis can occur at the same time for efficient protein expression. The reaction produces the protein of interest in just a few hours.
Emily Chen:
We've taken this process and combined it with our knowledge of producing high quality enzymes and proteins for over 50 years and translated that into a system that enables you to easily and efficiently express your proteins of interest cell-free. The NEBExpress Cell-free E. coli Protein Synthesis System was designed to be user-friendly. You have all that you need to express your protein of interest in three tubes, which contain the S30 synthesis extract, an optimized protein synthesis buffer, and T7 RNA polymerase.
We provide a standard protocol for a 50 microliter reaction, which can be altered to fit your specific protein of interest. NEBExpress is an open system, so you can directly manipulate the environment where protein production is taking place. RNAs inhibitor is provided with the kit to be used at your discretion. The volumes can be scaled up or down according to your needs. We have optimized and tested our system so that it synthesizes proteins consistently and reproducibly at different scales. All you have to do is mix the reagents together, add your own template, and incubate.
Aside from the kit components, only minimal equipment and bench space are required for the reaction. These are likely things already in your own lab. In fact, you can even perform these reactions at room temperature, eliminating the need for an incubator. This makes NEBExpress a great kit for startup laboratories or any scientists looking for analytical amounts of protein to give them quick results.
As we've mentioned, NEBExpress is a great system to save you time, space, and equipment. But what are the other benefits of NEB Express? This system is also designed for flexibility and compatibility with upstream and downstream workflows. You can start with a completely synthetic piece of DNA. You can perform assembly and amplification in vitro. NEBExpress is compatible with all in vitro workflows. There's no need to clone. NEBExpress components are also untagged, so you can add tags for easy affinity purification. The protocol makes it easy to get reproducible results with the same template, giving you less variability in expression. NEBExpress has also been proven to express a broad range of protein types and sizes. Be able to use one product for your expression needs. And without the use of live cells, there is no need to worry about cell viability or growth, which also allows you to express toxic proteins. Need more or less protein? Scale your reactions up or down accordingly for your experimental needs.
Though this webinar focuses on the NEBExpress Cell-free E. coli Protein Synthesis System, it's not the only cell-free option from NEB. We also offer the PURExpress in vitro protein synthesis kit. The main difference is that PURExpress is made with individually purified components, which are then reconstituted into a mixture, whereas NEBExpress is a cell extract-based system. As such, PURExpress is customizable with its components, which is great for specific types of experiments, including ribosome or mRNA display, and non-natural amino acid incorporation. NEBExpress is better suited towards workflows that require more protein or downstream his-tag purification. Both are designed to be user-friendly, have template flexibility, and minimal to no nucleus and protease activity.
So we've told you that you can use NEBExpress and get purified protein from DNA in one day, but how does this work? One option is to do this all in vitro with no cleanup steps. This can be done manually, but it is also 100% automation and multiplexing compatible. From an ordered piece of linear DNA, perform gene assembly with any builder, isothermal amplification with phi29-XT, cell-free protein expression with NEBExpress cell-free synthesis system. And finally, magnetic bead purification with our NEBExpress Nickel NTA magnetic beads. NEB has all that you need for this process.
Template flexibility and NEBExpress gives you more control over your workflow. Choose to use either linear or plasmid templates. The main requirements for either are shown here. Your template needs a T7 promoter, ribosome binding site, start and stop codons, an in-frame protein coding sequence, and a T7 terminator. All you need is your gene of interest that you wish to express. Then use available cloning or assembly tools and techniques to design and create your recombinant DNA. A controlled plasmid is provided in the kit that can be used as a cloning vector for your convenience. Your gene of interest can be cloned or assembled with an affinity tag, which allows for convenient purification. With assembly tools and quick expression at your disposal, you can test different tags, positions of a tag, linkers and cleavage sites and sequences. NEB has many options for purification of commonly used tags like His, MBP, and CBD tags. For example, we offer different site-specific proteases, as well as different beads and resins, some of which are listed here.
Paula Magnelli:
Remember, plasmid or linear DNA can be used as a template. Circular DNA, which is closed upon itself is very stable. However, it takes time to clone a gene into a plasmid, and this requires cell transformations and plasmid isolation. Linear DNA, on the other hand, is convenient because you can start your experiment directly from a piece of synthetic DNA amplified by PCR. And this DNA is then purified after a one-step cleanup. But linear DNA is more sensitive to degradation. It can be chewed from its end by exonucleases. You can use an exonuclease inhibitor, GamS, to boost the system performance.
Sometimes you might choose to uncouple the system. This means you separate transcription from translation. NEBExpress works very well with mRNA as template. You can prepare mRNA using HiScribe T7 high yield RNA kit using T7 RNA polymerase and nucleotides. To use mRNA as a template instead of DNA, you take a small aliquot of the high scribe reaction into the cell-free protein synthesis reaction. The mRNA will be translated to produce the protein, which you can see in this gel. The protein product is marked with the red dots. It is the band that does not appear in the negative control. Now that you identify the product of this reaction, you can see how they yield as good with mRNA as they wear with DNA template. This is because RNAs are very low in the NEBExpress protein expression system, making mRNA very stable. To summarize, you can use the HiScribe crude reaction directly into the NEBExpress reaction. But if you prefer, you can purify the mRNA first. Using the NEB Monarch RNA purification kit is optional. But this step allows you to quantitate the mRNA and control the input of template in your reaction.
The NEBExpress system contains all that's needed to express your protein. The user simply adds template. The results depend on the quality and quantity of this template. So I will take a moment to give you some tips to run a successful experiment when you start from plasmid DNA. Good quality plasmid preps will give you the best result. It is easy to get a good prep by following a couple of simple tips. When growing your cells in selective media, do not overgrow the culture. Do not vortex doing plasmid preparations. Keep your DNA in a buffer solution once you have isolated it and measure the concentration of your plasmid DNA.
Emily Chen:
With your template properly prepared, this system has been shown in-house to produce both eukaryotic and prokaryotic proteins over a broad range of sizes. Each lane shows the background proteins of the E. coli extract while bands marked with red dots show express target protein. Typically, you will get a yield of 0.5 mg/ml of soluble and active protein. When compared to other suppliers who offer their own cell-free protein synthesis kit, where expression here is indicated by a blue dot, see that NEBExpress has comparable or increased target protein expression. Further, NEBExpress reactions can be run directly on SDS-PAGE or used immediately for other downstream applications due to buffer compatibility. It is formulated without crowding agent, and so there is no need to buffer exchange beforehand, which is not the case for other cell-free products.
NEBExpress is also powerful enough to express multiple proteins at once. It is capable of synthesizing two or more proteins within a single reaction. For example, adding bGal and BglA plasmids results in expression of both. Depending on your needs, you can add more templates as seen in the other lanes. Co-expression of proteins in NEBExpress is much easier than doing it in live cells. Why? This is because cells, no matter how much you try to control them, will prefer to make one protein over another. It is often difficult to get the ratios you want. And each new batch of expression may give you variable amounts. As a result, co-expression in vivo often yield only a fraction of the right product and a lot of side products that aren't useful. In comparison, cell-free is much more responsive to template input. The system will make proteins in whichever ratio you want just by changing the input of each DNA. You can expect the same results each time as this can be done reproducibly in vitro.
NEBExpress reactions can be incubated at different temperatures depending on your expression needs. For short incubations, targets are quickly made at anywhere from 30 to 42 degrees Celsius. However, NEBExpress can also sustain synthesis for an extended period of time at low temperatures, which can increase final yields. Expression at low temperatures can also aid in the synthesis of more sensitive targets to help promote proper folding, solubility and stability. Even after a day at room temperature, there are no signs of proteolysis, which is by design due to how the E. coli strain was engineered. So depending on the protein and your goals, you can quickly check the best conditions for your target.
Some proteins, especially those containing multiple disulfide bonds, need more help to fold correctly than just lower expression temperatures. This class of protein includes many secreted proteins and proteins of therapeutic interest. In these cases, we highly recommend using a companion product, the PURExpress Disulfide Bond Enhancer, NEB #E6820S. Adding the disulfide bond enhancer at the beginning of your reaction promotes proper disulfide bond formation by assisting with the oxidation of cysteine thiol and to correct misoxidized substrates. Then with proper folding, you can increase the amount of active protein. You can see this in the chitinase activity assay where the protein is much more active with the disulfide bond enhancer added. This is a product that can be used for both PURExpress and NEBExpress.
Depending on your downstream goals, you may need to purify your protein target. This can be done manually, but there are also strategies that are more friendly to automation than others. For instance, spin columns are not scalable or friendly to multiplexing while magnetic beads are. Here's an example of a purification scheme after cell-free expression. You can bind your protein to magnetic beads that have an affinity for your specific tag. And this can be done while the protein is being made, which saves processing time. After washing away impurities, you elute your protein from the beads. This can be easily adapted to different volumes, plate formats, or used in an automated workflow.
The simplicity of the reaction setup makes the system well suited for scaling up, either by hand or with automation. A master mix can be prepared up to an hour before use when stored on ice. Then this can be aliquoted in whatever desired volume or format that you choose. At New England BioLabs, we tested the different possibilities with NEBExpress through our application notes, which can be found on the product page online. In this particular app note, we demonstrated automated cell-free expression and purification, essentially using the purification scheme shown two slides ago. Just set up your reactions and come back to purified protein.
You can see here that proteins of a range of sizes could be expressed using a master mix and automation in the gel on the left. And on the right is the end result of purified protein. These reactions were set up throughout an entire 96-well plate. The results here demonstrate reproducible expression with the same performance across columns and rows. Aside from the stellar expression and purification, we show that a master mix can be formulated and then dispensed without a drop in performance. And also that magnetic beads can be dispensed evenly and handled by automation. In conclusion, we show with minimal hands-on time and little intervention, one could obtain multiple pure samples in a matter of hours. This is great for workflow efficiency and enables high-throughput screening.
Paula Magnelli:
In this application note, our colleagues showed that NEBExpress can be dispensed with an acoustic liquid handler. This is very helpful for scaling down to minimal volumes down to a few microliters. They show that it's possible to run very small protein synthesis reactions. This application is great for high-throughput screens where you need to make the most of your reagent supply. And the details and the text of this app note, you can find it in our products webpage.
This is another application note showing a complete workflow that is 100% in vitro. This means no cells involved at any step. There was no gene cloning. Synthetic linear DNA modules were assembled into a circular DNA structure by Golden Gate assembly using any bridge. To obtain enough template, the circular DNA was amplified by rolling circle amplification or RCA using phi29-XT RCA kit. The DNA product was then used directly from the RCA reaction as template for protein production using NEBExpress. I want to highlight how cell-free proteins expression comes together with other technologies to enable completely new pathways for protein expression. You can go from a small amount of linear DNA to purified protein in one day, but this is not just about saving time. A complete in vitro workflow lets you test proteins that are artificial and natural or unclonable.
The last example I will show today uses the NEBExpress Protein Synthesis System to make antibody fragments. Antibody fragments are growing in popularity because they are easier to produce and they are easier to assay in comparison to full length antibodies. In this example, my colleagues prepare two types of antibody fragments, scFv and VHH. Using cell-free expression, you can find binding variants and optimize your candidates through successive rounds of expression in a matter of days. The same is true with the production of active scFv variants. The antibody fragments expressed in vitro had similar binding affinities compared with fragments produced in vivo. The fragments were tested directly from the cell-free reaction. No need to purify before the binding assays.
Emily Chen:
In summary, NEBExpress and automation let you express, purify, and test within a single day for faster results. NEBExpress Cell-Free Protein Synthesis is your tool for success. Thank you for joining us today. Please feel free to ask us questions about the NEBExpress Cell-free E. coli Protein Synthesis System. Hi. So for our Q&A, we have some questions here. So one of them is I've tried NEBExpress, my protein just doesn't express well. What things can I do to try and optimize my expression? I'm also going to combine that with another question that was asked, which is how long should I let my proteins express for? So there are a few things. One of the more important things is checking your template, making sure it has the required elements that we mentioned. You can also increase your expression by codon optimization for expression in E. coli. You can also try different incubation temperatures and times. This will all depend on your template. And you also want to make sure that your reaction has plenty of space for variations. And so a lot of this optimization is template dependent. And so you can try these different things really quickly to make sure that you're getting your optimal conditions for your expression.
Paula Magnelli:
Hi, hello. I have here also two questions that I'm going to combine in one which were questions about producing membrane proteins in the system or proteins that are glycoproteins or COVID spike proteins and more complex eukaryotic proteins. And the question is first, the answer is first, this is both are bacterial. The systems presented today, they are of bacterial origin. There's no glycosylation. And as we explained there, there is a possibility to have disulfide bone formation when adding the disulfide bond enhancer. And there is the possibility of finding the right condition of optimization to produce complex proteins, protein dimerization and different things. But this is not a native eukaryotic system. And in terms of the production of membrane proteins, oftentimes this requires the addition of lipids or other substrates for the membrane proteins to assemble. So that is described in the literature, but this is not something we have tried yet here and therefore we didn't present an app note about that.
Emily Chen:
Someone also asked to repeat the general output of protein that's possible to generate. So typically with these reactions, you can expect yields of 0.5 mg/ml.
Paula Magnelli:
Here I'm going to combine a couple also questions about the promoter. When the sequence is under a different promoter, say you have a plasmid that is not under the expression cassette, it's not under the T7, the T7 RNA polymerase promoter, one possibility is to prepare the mRNA offline, uncouple the reaction with, for instance, an SP6 HiScribe kit instead of the T7 kit. So if you have an SP6 promoter, you can transcribe your mRNA using a kit or the corresponding mRNA polymerase and use then that reaction to feed into the cell-free protein synthesis reaction. And in terms, there's another question speaking of the promoter, how close to the edge of the promoter, the DNA cassette has to start? And we recommend having a few extra nucleotides downstream the, six nucleotides downstream the beginning of the T7 RNAs polymerase promoter. But we also have tested starting right at the edge and it works as well.
Emily Chen:
Somebody also asked if we provide the disulfide enhancer separately. So yes, this is under the NEB catalog number, E6820S. Somebody also asked if it's possible to add co-factors to the synthesis reaction if the protein needs it. And this is yes. So you can directly add whatever you need to the system. It's an open system so you can manipulate your environment.
Paula Magnelli:
Here I have a question about yields in NEBExpress comparing plasmid DNA and linear template. And yes, there is a difference in yields. The yields using linear template are lower than the yields using plasmids. Nevertheless, particularly when you add the GamS additive, there is a pretty significant expressions, and it also depends on how you construct that linear template. It is important to have a complete T7 terminator, which prevents nuclease regulation and have the right confirmation, the right 5′ UTR sequence.
Emily Chen:
We have a question about, can you expect endogenous enzymes of E. coli to be active specifically methionine deformylase and methionine aminopeptidase, which often together removes internal methionine? So endogenous proteins will be active, though when you're overexpressing your target, it will likely overwhelm this process, so you'll typically end up with a mixture of processed and unprocessed.
Paula Magnelli:
Hi, I see another question about maximizing the output for expressing membrane proteins and other difficult-to-express proteins. Back to membrane proteins, remember that it is important in those cases to include polymers and additives where that membrane protein can correctly assemble and fold. And that depends on the application, depends on the type of protein. But in general, for difficult proteins that you anticipate challenges because they are complex, they have difficult foldings, usually reducing the temperature, tuning the input of template, sometimes less is more, increasing the time, or a combination of all of those can lead to a total ratio of soluble protein and correctly folded protein to compare with an suboptimal condition.
Emily Chen:
All right. Can we use the system to incorporate a natural amino acid? For example, amino acids in quick reaction functional group. Typically for incorporating a natural amino acid, we more recommend a PURExpress kit as it's minimal and defined. So if that's something you're interested in, definitely look into that kit.
Paula Magnelli:
Okay. I have a question here about proteases, and someone is asking what proteases are active and if those proteases will be active enough to activate a protein that requires those. So this system to start with has a very, very low general protease activity. So we have not tested how active certain specific proteases are, but I would suggest that if a protease is necessary for a specific cleavage for activation, maybe the best route is to add it at the end of the reaction if you don't see the activation and the cleavage that you are expecting.
Speaker 3:
Well, we are coming up on time. If we didn't have a chance to get to your question today, we will be sure to follow up with you offline. And if you think of any other additional questions you'd like to ask, feel free to reach out to us at webinars@neb.com. The webinar will also be available on demand to watch or share with your colleagues. Thank you so much, Emily and Paula, and thank you all for joining in today. Hope you have a wonderful rest of your day.
