HaloTag as an expression partner – Author Interview

We recently published “HaloTag is an effective expression and solubilisation fusion partner for a range of fibroblast growth factors”. In this study, authors Changye Sun, Yong Li, David G. Fernig and their colleagues examined the use of HaloTag as a fusion protein.

Changye, Yong and David comment on their research and experience publishing with us.

PJ: Can you tell us a bit about yourself?

Changye Sun using the red fluorescence dye labelled Halo-FGF1 to do cell imaging work

CS: I (Changye Sun) am doing my PhD supervised by David Fernig in the Department of Biochemistry at the University of Liverpool. Our group is working on structural biology, which covers the protein-heparin/heparan sulfate (HS) interaction and the study of extracellular matrix (ECM) structures, and nano-biology, which applies nanoparticles to some biological applications.

Yong Li

YL: I am currently a PhD student in Dave Fernig’s group. The project I have spent almost four years on is to use the phylogenetic relationship of the FGFs to understand the structural basis of the specificity of the interaction between FGFs and HS. Since my project focuses on the FGF family, I need to express and purify as many as possible. The work on our paper, which shows that using HaloTag markedly increases the expression and solubility of FGFs, has made a huge difference to the number of FGFs I can study. The most surprising advantage of HaloTag is its ability to solubilise these proteins which are still insoluble even when I tried many different culture conditions and solubilisation tags, like Sumo. If anyone likes our research and has similar problem with their proteins, I do recommend having a try with HaloTag. Since we published our research last week, it had been downloaded many times from PeerJ and my Research Gate account. I have already suggested to my colleagues that PeerJ is a great choice if they want to publish their work as open access.

David Fernig

DF: I’m a biochemist, though I do many things – training is one thing, research another and in the end there is just science, engineering and mathematics, and we touch on all aspects depending on what our needs are. Liverpool is a very collegiate university, so if the path leads into an area where you know nothing, a short walk on campus always brings you to an open door and a colleague who is an expert. My glass is always half full, even when empty :).

PJ: Can you tell us a bit about your team?

DF: The team? All graduate students, doing great work and fantastic fun.

PJ: Can you briefly explain the research you published in PeerJ?

CS: Our publication shows how to efficiently produce soluble fibroblast growth factors (FGFs), which were difficult to express as soluble proteins. The produced FGFs were tested for their biological activities and the results suggested that the produced proteins with the HaloTag gave similar activities to the proteins without a tag.

PJ: Do you have any anecdotes about this research?

CS: Yes. Actually, the HaloTag was used to covalently label Hypoxia-inducible factors for imaging (see Sarah, another author). Because FGF2 is a better characterized protein, which we can make recombinantly, and it binds to heparin, we brought the HaloTag to our lab and cloned it into the plasmid containing FGF2. When we tried to express and purify Halo-FGF2, we found the expression was very large and a lot of Halo-FGF2 was in the pellet. So, we thought the HaloTag could increase the protein yield and a lower temperature was required for expression. Then, we had a meeting and discussed this phenomenon. We thought that as HaloTag is a large and soluble protein, the FGF would be expressed more slowly and be folded better with the HaloTag. So, the HaloTag might be good soluble fusion tag for protein expression. Then, we moved HaloTag onto the other FGFs and we got the nice results, as shown in our paper.

PJ: Can you briefly explain the research you published in PeerJ & Do you have any anecdotes about this research?

DF: An accident, though there is no such thing as luck, a prepared mind and experience pay dividends. We needed a good positive control for Sarah’s work on HIF, so we volunteered FGF-2: we have decades of experience, purified it from bovine pituitary in the 1980s and were one of the early groups to make recombinant protein. Changye and Yong came into my office saying that Halo-FGF2 didn’t express well, because there was a lot of protein in the insoluble fraction. My reaction? “Wow, that is brilliant”. After explaining why full-length FGF2 expresses relatively poorly and is always soluble, my interpretation that we had increased expression was accepted as a possibility. It was at this point that we planned the rest of the work and the paper, and as each FGF that we had failed previously to express as soluble protein came out in the soluble fraction, my reaction was vindicated. However, HaloTag is not a universal panacea for expression problems, but the more solutions you have, the greater your chance of success.

PJ: What surprised you the most with these results?

CS: We used to think it’s very difficult to express and purify some problem FGFs, such as FGF6, FGF17 and FGF20, but when the HaloTag worked, everything in the expression and purification had become easier and the biological activities and HS binding are still the same.

DF: That we couldn’t get a stimulation of mitogen-activated protein kinase phosphorylation with FGF16 and FGF17 (we tried!), but we could get a stimulation of cell division.

PJ: What kinds of lessons do you hope the public takes away from the research?

CS: More studies on the FGFs’ biological and biomedical functions would be explored, which means more protein medicines related FGFs would be developed in the future, and this might help us to treat some serious diseases.

DF: That we know very little about biology, health and disease: most research is on a small number of molecules, while the body makes use of hundreds of thousands of molecules. We need to spread research resources across the breadth of biochemistry and physiology.

PJ: Where do you hope to go from here?

CS: Thanks to the HaloTag, we had successfully purified some stable and highly expressed FGFs which had been applied to my project, the binding and dynamics of FGFs in extracellular matrix, and Yong’s, heparin/HS specificity of FGFs. There is still much more work to do with these proteins. For example, some FGFs, such as FGF6 and FGF17 structures will be studies and lots of imaging work will be done as well.

DF: C elegans makes do with just two FGFs. Armed with almost a full set of human FGFs we can begin to answer a simple question: why are there so many, what selective advantage(s) is given by a family of 22 proteins?

PJ: If you had unlimited resources (money, lab equipment, trained personnel, participants, etc.), what study would you run?

CS: Translational medicine! We’ll make more stable FGF and explore their medical applications from their structures and cell activity to their functions in tissue/organ and body.

DF: I would give most of it back, big labs don’t deliver, or I would spread it around to 1,000s of PIs! For my own part, I would give a colleague the funds to build a bespoke light sheet microscope (we have a Zeiss one, which is excellent, but home-built gives you flexibility) and we would measure FGF function in organoids with a view to developing FGF-based therapeutics.

PJ: How did you first hear about PeerJ, and what persuaded you to submit to us?

CS: I found this from my supervisor, Dave. Because PeerJ is an open access journal, people who are doing related research can easily get our results for free, which would be a good thing for both our citations and the peer researchers.

DF: Not sure, it was sufficiently long ago that the professorial memory has forgotten! This is my third paper, as part of my push to open access and open data.

PJ: Do you have any anecdotes about your overall experience with us? Anything surprising?

CS: At the beginning, I thought PeerJ was a too young a journal, which would accept any paper that had been submitted, but from my submission and the paper citation history, I find PeerJ is a revolutionary way for publication and I trust it will be much better and stronger in the near future.

DF: We won’t let Changye forget that during the initial submission he assiduously copy/pasted in the e-mail addresses of all authors, except for his own, as he knows it: this he typed and introduced a typo, which needed correcting!

More seriously, converting the figures into publication format at the start and pasting in the legends, etc., seems like work, but it pays in the long run. I have also started to make use of the preprint server, as this gives us priority.

PJ: How would you describe your experience of our submission/review process?

CS: Actually, this is my first publication. And Dave took me through all the submission and review process. I found it was very clear and easy to submit a paper to PeerJ, but the reviews were very strict, which suggests the referees of PeerJ select papers very carefully. P.s, the edition is very nice.

DF: Smooth. Also most welcome is the fact that the deadline on re-submission is a guideline. This is realism. A research team cannot always do the required work in X days, as enforced strictly by quite a few journals. If the reviewers’ critiques are to be addressed properly, this can take time.

PJ: Did you get any comments from your colleagues about your publication with PeerJ?

CS: They said it’s a nice publication. We shared the most difficult work in our research and gave many results. This is also good for my PhD viva, because a paper is very convincing.

DF: I think they fall into two groups: those still wedded to impact factor and those who are moving fast into open access and open data. I may be embarrassing the first and the second are probably ahead of me and wondering what took me so long!

PJ: Would you submit again, and would you recommend that your colleagues submit?

CS: Definitely! I had told many friends about PeerJ and one of my friends passed this message to his supervisor and they published their paper, “Caveolar disruption causes contraction of rat femoral arteries via reduced basal NO release and subsequent closure of BKCa channels“, even earlier than us – lol.

DF: Yes – there are quite a few papers from the institute of Integrative Biology in PeerJ and there will be more. I keep pestering our library to take out an institutional “subscription”….

PJ: Anything else you would like to talk about?

CS: We would like to make more contributions to open access publications and hope more people can get involved and fight for publishing papers with cheap prices and open access, especially for the young researchers who are not in a position to pay for the expensive review and open access option.

PJ: In conclusion, how would you describe PeerJ in three words?

CS: Open Smart Beauty

DF: Hard, but fair

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