Interviewers: Lydia Morrison, Marketing Communications Writer & Podcast Host, New England Biolabs, Inc.
Interviewees: Mehmet Berkmen, Senior Scientist, NEB Protein Expression & Modification Research; Paul Riggs, Retired Senior Scientist, NEB Protein Expression & Modification Research
Lydia M: Hello and welcome to the Lessons from Lab and Life podcast. I'm your host, Lydia Morrison, and I hope that our podcast offers you some new perspective. Today our podcast is focused on the lessons that New England Biolabs has learned about protein expression. NEB has been offering purified restriction enzymes since the mid 1970s and it quickly became clear that in order to meet the research community's needs we needed to be overexpressing and purifying these enzymes. I'm joined today by two dedicated NEB scientists, senior scientists Mehmet Berkmen, better known to his friends and colleagues as Memo, and the recently retired and much celebrated Paul Riggs. Thank you both so much for being here today.
Paul R: Thanks for having us.
Mehmet B: Thank you very much.
Lydia M: I was wondering, Memo, if you could tell us how long NEB's been involved with protein expression?
Mehmet B: NEB's been involved in protein expression from its inception onwards. It was a company that was founded in 1974 to make proteins that research scientists wanted. And the tool that we use is E.coli. And we use E.coli to make all our proteins, we make currently over 550 different proteins from all domains of life, from archaea, eukaryotes, plants, you name it, even viruses. And we make all these proteins using E.coli. About 90% of our proteins are expressed in E.coli, so you could say that NEB is an E.coli expressed enzyme company.
Lydia M: Oh, that's good. I like that. How has NEB used protein expression in research?
Paul R: So in the beginning we were making our enzymes from their native sources, but it quickly became apparent that to make more it would be a good idea to clone the enzymes and get overexpression. So that was a trendsetter for the industry, nobody had cloned restriction enzymes before that. Later on we started some basic research in parasitology and there we were cloning proteins in order to study them and possibly make a vaccine from proteins made.
Paul R: So for the enzymes that we sell, in the beginning the overexpression we got was just by cloning the enzyme and putting it into E.coli. As time went on, we did a lot of enzymes that way and we had huge levels of overexpression, a hundred fold or more, just by cloning the enzyme and putting it in a different host. Later on we started to realize that a little bit more sophisticated positioning of promoters and control elements could lead to even better expression. And over the years we've ended up doing that with most of our products.
Lydia M: And how did that change the availability of these reagents for researchers?
Paul R: Reduced the cost quite a bit and made it so that we didn't get in a treadmill of having to make the same protein every few weeks. So it made a huge difference for both the market, for people buying the enzymes, and for us.
Lydia M: How did we go about using protein expression for our own product development?
Paul R: Well, at the same time as you realize that you can make large amounts of proteins, you can then use those large amounts to study them. And so we became the company that knew the most about the enzymes that we sold and we started to collaborate with some crystallographers to do some crystal structures and we did a lot of basic research on just how the enzymes worked.
Lydia M: And when was the first NEB protein expression product made available to customers?
Paul R: Oh, that was around 1990. We had some applied research at using a fusion tag to simplify the process of getting overexpression and purifying the protein. That was the protein fusion and purification system, which uses the maltose binding protein as an affinity and solubility tag. That was actually what I came here to start working on under Chudi Guan, who was the one who invented that system.
Lydia M: And you first came to NEB as a post-doc?
Paul R: I was his post-doc, came in 1987 and he went back to China, so I got hired to continue his work.
Lydia M: Well, we're lucky to have had you for 32 years. So what were the first protein expression systems that NEB used?
Paul R: So in the beginning we used just simple vectors that were around, pBR-322 and pUC. And just by cloning them onto those vectors, we would get decent overexpression. But as time went on and as our product line expanded into some proteins that you might need in larger quantities, we found that using variants of the lac promoter, the T7 promoter and specific control elements to control expression gave better results and allowed some of the clones to be more stable so we could grow them in large amounts.
Lydia M: And when did we start to incorporate fusion proteins and tags into our overexpression?
Paul R: The first fusion proteins were made mostly to simplify the purification and more as a tool for researchers than a tool that we used a lot at NEB. Although we did use them for some proteins. Later on it, as the field developed, we realized that things like the solubility enhancing properties of maltose binding protein would actually enable us to make some proteins that we normally couldn't make in E.coli and they started to be more widely used in-house.
Lydia M: And what about the prominence of tags? Did we start using tags for easier purification or for our own internal research?
Paul R: Both. Really both. Because we were selling them to researchers who needed those aids in purification, but also we were doing basic research here that the tags helped a lot. The IMPACT system allowed you to basically do a one column purification where you could get a lot of protein really fast, and that facilitated the research by giving you an easy way to make something as soon as you could identify the clone.
Lydia M: Absolutely. Memo, can you speak to what some of the advances are that we've seen in protein expression over the years?
Mehmet B: Surely. I'd like to begin by saying that one of the ways we can define NEB is that we're a company by scientists, for scientists. Everyone who works here is a scientist, so we're constantly reading the latest and want to incorporate the latest developments in protein expression. And what has happened over the years is that as the depth of our understanding of protein expression has increased, the capacity to express weird proteins from eukaryotes and viruses have also increased. We now want to express cofactors and specific chaperones that enable these weird proteins to be expressed in E.coli. We have genetically engineered various E.coli strains to be able to do you eukaryotic biological processes that normally E.coli could not do. We use different expression systems, different compartments, different gene expression methods, and the advances in the field are constantly followed and applied at NEB daily.
Lydia M: That makes a lot of sense. What are some of the challenges that affect protein expression?
Mehmet B: Well, the biggest challenge is that E.coli is a bacteria that grows in our guts, so it has over the last few billion years evolved to live and defend itself in the gut. When you take a eukaryotic protein from a plant and you put it into a bacteria, it does not have the genetic knowledge to express this weird protein it has never seen. So what we have to do is now take those information from the native host and genetically incorporate it into bacteria and make E.coli do something it has never done in billions of years.
Lydia M: So how do we address all these challenges?
Mehmet B: By doing basic, good research. The strength of NEB over the decades has been that we always follow product development by following the science. So when we do good science, you kind of do the same question/answer method that any academic lab does. So if you try to clone a protein, it doesn't express in E.coli, you have a massive challenge and you have to do good science to address why it's not folding. And your aim isn't to make the product, but to understand the biology of that enzyme.
Lydia M: So you're saying that because NEB scientists are, I think, lots of them, academic scientists at heart, and we do a lot of in-house basic research here, that we really sort of understand those customer needs and really the requirements and how quickly the requirements change in a specific field, that we're able to sort of try to keep up with that.
Mehmet B: Yeah, I mean you have to think about it. This company was founded in 1974 and the research and the science since then has changed exponentially and we have to keep up to that. But it's not a matter of, "Oh, the science is changing, we have to catch up." But it's a matter of being at the forefront of science and creating those new developments. So a lot of the protein expression developments are first heard in the world through the basic research done at NEB.
Lydia M: That's amazing. Do you have an example of where basic research was impacted by protein expression?
Mehmet B: Definitely. One of the classic examples is inteins. These are self-splicing proteins and it was discovered through a basic research at NEB and it was a very big puzzle, how this protein was constantly splicing itself, and we had to do a lot of effort to understand this biology. And that resulted in not only uncovering biological processes that the research field use, but also allowed us to develop protein purification methods. My colleague Paul was just talking a few minutes ago about single step purification through a column, and that was all based on intein technology, which basically is you fuse your protein of interest to another protein that binds to a column, and through just adding a chemical reagent it will self-splice itself and come out of the column. So you don't have to do any fancy purification methods. And this all came about by doing basic research.
Lydia M: Wow. That is truly amazing. What are some of the challenges that affect protein expression?
Mehmet B: Historically, if you take a look at the back, the first days of NEB, we were basically expressing restriction enzymes, which is a bacterial system. Cloning and expressing those proteins in bacteria were not that challenging, but as our product portfolio expanded, we went into proteins that are expressed from plants, archaea, and especially eukaryotes. And those proteins require a lot of cofactors and a lot of systems that bacteria don't have. Generally speaking, expressing eukaryotic proteins in bacteria is quite a challenge and you have to bring in the biological systems in eukaryotes into bacteria. But other protein groups that are very challenging are membrane proteins, which tend to be very difficult to be overexpressed. Luckily we don't have many, or any, membrane protein products that we sell. Toxic proteins is generally a big problem at NEB even today. And that's because a lot of the enzymes we sell, modify and change in nucleotides and those tend to be toxic to bacteria. And that's a big, big challenge and we're constantly addressing that.
Mehmet B: Another big class of proteins that we have difficulty expressing are those that require protein modifications after they're translated, such as disulfide bonds. And we have engineered special strains to address that, but there is also lipidation and glycosylation and other processes that bacteria cannot do. And the solution we kind of think of is to understand the processes that modify those proteins and bring them into bacteria genetically.
Lydia M: So we would clone those scaffolding proteins or proteins that affect glycosylation or something like that, we would clone those into the same bacterial system for expression?
Mehmet B: Yeah, you're hitting on the biggest challenge of protein expression right there, which is that for every protein you need to express, you really need to have a deep understanding of that protein. Its biology, how it's expressed, how it's used. And only when you have a good understanding of that protein and its role can you solve the solution of protein folding. But the big challenge is that for every protein you have to do this process again and again. Each protein is unique in its own expression portfolio and its biological problems. So deep understanding of the biology of the protein is essential for good high yields.
Lydia M: Absolutely. And do you find that the groups of proteins that are necessary to accurately express and fold a protein, do those apply to maybe more than one protein in a certain family, or is it really a unique problem every single time?
Mehmet B: My personal opinion is it is a unique problem every certain time. Even when you change a single residue, you find that the protein expression portfolio changes a lot. We have very few general solutions for protein classes, we tend to think that anything above 50% solution is great success. Do you have anything to say, Paul?
Paul R: The most general thing that works for bacteria is to slow down the translation, because the translation in bacteria is inherently a lot faster than most of the eukaryotic proteins do. So by lowering temperature and slowing down translation we can make a lot of proteins that you can't normally make in E.coli. When it comes to folding, as Memo says, it's really a protein by protein approach. Some of the things like disulfide bonds is a little bit more general and you can usually tell by looking at the protein or knowing a little bit about the protein in its native source that that's going to be a problem. And then you can apply a strain such as SHuffle, which is made to help form those disulfide bonds. So it really depends on where the problem in folding comes from.
Lydia M: So the problem with the translation rate in E.coli or in bacteria, does that increased rate of translation mean that you're increasing potentially the rate of the errors that are incorporated? Is that the problem? Or is it more of a folding problem?
Paul R: I think it's more of a folding problem. When the protein sort of folds as it comes out of the ribosome and what the model is that if you make the protein too fast, areas of the protein that don't normally interact have a chance to interact and misfold.
Lydia M: I see, I see.
Mehmet B: If I can add to that, we currently believe that transcription and translation are coupled. So as you make the RNA, you also at the same time make the protein. And the rate of RNA synthesis in bacteria is much faster, so the RNA tends to be naked and fold on itself. So the ribosome has also difficulty of access. So the process, as I was saying a few minutes before, you really have to understand the biology of everything to address the scientific problem of how to make a protein.
Lydia M: So how do we address these challenges?
Mehmet B: By doing good basic research. We have the luxury at NEB that we don't have the usual pressures that an academic lab has. We have amazing resources in this building, both financially and scientifically, so that gives us the space to address these very difficult questions over a long time. We have the patience at NEB to put due diligence and smart minds at a difficult problem. And to our success, we usually end up solving it, and it may take years in some case. We have an enzyme that we just released this year that took a team of scientists several years just to express.
Lydia M: We're certainly lucky to have such dedicated scientists and long-term vision at NEB; what protein expression approaches are we using today?
Paul R: I think we use a lot of tight control expression vectors. What we find is when you scale up, you often have a problem with clones being unstable. And so standard T7 kind of expression works for a large subset of proteins, but sometimes you have to get a little bit more creative when you're growing large amounts. We use some solubility tags and affinity tags, mostly maltose binding protein, and the IMPACT system for particular products that really perform well in that situation.
Lydia M: You've mentioned the IMPACT system a couple of times; what does the IMPACT system involve?
Paul R: The IMPACT system is the one that uses the intein technology so that you bind the fusion protein to the column and then you add a reducing agent which initiates the cleavage of your protein and your protein comes out of the column in one step, clean.
Lydia M: So is that a single use column?
Paul R: You can recycle the column a number of times because you can strip the column afterwards.
Lydia M: What advancements in protein expression would you like to see in the future?
Mehmet B: I think with the rise of synthetic biology, the demand for high throughput protein expression is growing. What that means is that people would like to test hundreds if not thousands of different protein variants in one day, in a few hours, and decide which protein variant to further their studies. What that means is E.coli currently, the way standard a cloning, ligation, transformation, wait next day, grow your bacteria to a liter and then purify your protein, is not applicable to the demands of synthetic biology. The solution is cell-free protein expression. You now can express a protein by simply putting a piece of DNA into a solution which has all the protein expression components in it. It has all the transcription and translation machinery and all you have to do is add a piece of DNA and you will have a protein in a test tube within minutes, or at most an hour. The problem with this is that scale up is currently very challenging. You can do these experiments at microliter scales, perhaps at milliliter, but you cannot have milligrams or grams of protein produced using this method. And I think this will be the new challenge for protein expression.
Lydia M: It's amazing to hear how far protein expression has come in the last 37 years. We've gone from expressing protein in the original organisms to being able to express large quantities of protein and now to being able to express large quantities of protein without even using a cell. So I just want to say thank you to you both so much for your research and dedication, especially to Paul for being part of those original forward-thinking folks to be able to scale up restriction enzyme production and really being able to bring affordable reagents to the hands of researchers all over the world.
Lydia M: Thank you both so much for being here today.
Paul R: It was a pleasure.
Mehmet B: Thank you for inviting us.
Lydia M: Thanks for listening to this episode of our podcast. You should definitely tune into our next episode when I'll be joined by the inspirational doctor Eva Nogales, who is a Howard Hughes Medical Institute investigator, as well as the head of the Division of Biochemistry, Biophysics, and Structural Biology of the Department of Molecular and Cell Biology at the University of California, Berkeley. Dr Nogales will share with us an overview of some of her work in the field of cryo-electron microscopy, as well as some life advice for anyone considering a career in science.
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