Keep calm and express your proteins

If you have ever worked with proteins you know the struggle. You identify your gene of interest, design the perfect construct, send it out to be synthesized and wait for it to be delivered to you. After some weeks you receive it, insert it into your host cell and ta-dah! Your protein is not expressed like you expected. Is it toxic? Insoluble? Not expressed at all? Or maybe just at very low levels that don’t meet your requirements? Time to troubleshoot.

If synthesising one protein in lab conditions is hard, imagine the challenge when your production goals are at an industrial scale. But, why are proteins such a big market? When and how do you start expressing a protein of interest at an industrial level? What are the best systems to do so? What is the most cost-effective option? And finally, how can the production be improved?

To get some insights about all these questions of the protein industry today we talk to Juergen Mairhofer, protein expert, co-founder of enGenes Biotech and partner of Rafts4Biotech.

When asked about the industrial relevance of proteins, he has no doubts: “Proteins are everywhere! Its starts from medicine and then it reaches out to industrial enzymes that are inside your washing powder and then it goes even further in the next generation of products will be likely meat substitutes or even textile fabrics. It’s such a big market!” Thus, no sector is free from these fascinating molecules.

But the first limitation that both industries and researchers face when starting to produce a protein is obtaining the desired DNA vector with the gene of interest. “I think that in the near future, every lab will have a benchtop DNA synthesiser, like the one developed by Craig Venter together with Synthetic Genomics Inc. (SGI). Within a few hours you will have a DNA construct or even whole libraries synthesized and you can start cloning! This way you won’t have to wait like nowadays, depending on the size of the gene you wait 4 to 6 weeks or even longer”. But until this technological dream comes true, we will have to keep waiting for our DNA to be ready to start our protein expression challenge!

Once you have obtained your protein, the next step will be to meet the high demands of the markets. To do so you will have to optimise the yields choosing the system that better fits our needs. Mairhofer points out two expression systems that are widely used in biotechnology: “On one hand mammalian systems like CHO cell lines or HEK cells are used for biopharmaceuticals, because for complex proteins like antibodies or virus like particles that you use in cancer therapy or for vaccination you need human glycosylation patterns. And that is something you can only achieve with mammalian cell lines.”

The downside to this approach is the price, as it requires large amount of expensive media to cultivate the cells. So, if the requirements aren’t that specific, microbial expression systems are the way to go according to Juergen: “The picture drastically changes when you use E. coli or yeast because the media formulation is much cheaper and process times are much shorter, therefore you can produce proteins fast and in a very cost-effective way. So as long as you don’t need the glycosylation pattern for therapeutic applications most companies go for E. coli because it is the cheapest option and a well proven technology that is on the market for more than 30 years.”

But as you know so well, working with living organisms can be challenging in a lab and when taken to an industrial setting, these challenges can compromise the viability of a biotechnological application. Cells are naturally excellent protein producers, but getting them to deliver high amounts of a specific proteins can be hard. They will always preferentially invest their resources in producing the enzymes that they require for their own growth and survival.

To overcome this limitation, Juergen and his team have decoupled biomass growth and recombinant protein production in E. coli, successfully redirecting all the resources to the obtainment of the protein of interest: “We can shut down the host RNA polymerase so that the cell is no longer able to produce its own mRNAs. At the same time, we have a phage derived T7 RNA polymerase that is not affected by the inhibitor protein that we express and that massively over-transcribes the gene of interest. Finally, we have a good proportion of the mRNA of the gene of interest that is then exclusively translated by the host cell machinery to recombinant protein and since there are no other mRNAs around from the host, we really can channel the metabolic resources into the recombinant protein and thereby we get really good yields.”, explains Mairhofer.

Another burden for the manufacturing of proteins is that they can be toxic and interfere with the many other metabolic reactions that happen within the cell factories. To solve this problem, at enGenes they keep exploring alternative options to improve protein expression, like the ones offered by our favourite cellular compartment: lipid rafts: “Rafts are great tools to bring together different proteins that need to be in direct contact or near to each other” In this context, the rafts serve as specialized compartments that can also reduce the interferences with the rest of the metabolisms of the host cell. But it is not as easy as it sounds “From the protein expression perspective, it is a very interesting project for us because we need to express not one but multiple enzymes at the same time and in a very specific ratio further increasing the challenge”.

So, if you are stuck with the expression of a protein, don’t lose hope. Protein experts are using synthetic biology to design the perfect microbial bioreactor to help you.