So How Do You Make a Protein?

Nepenthes x ventrata 'Monkey Cups'

I realised some time ago that I - me as a human - I need to make things. It is a fundamental part of who I am. Seeing things that I’ve made confirms to me that I am alive, functioning and having an influence on the fabric of the world. That thing exists because of me. And it can be anything - drawings, paper houses, files with organised notes in. When I’m feeling ignored or insignificant, making things - generating things - lets me know that I matter.

You might say that my function is to make things, though really I’m a lot more complex than that.

The truth is that all of the bits of me - each one of my cells - is making things all the time, and that’s not special to me. All living things, and at their base their cells, need to constantly make and replace proteins, lipids, and carbohydrates to function, to generate energy - to live.

All cells are hard-wired to do this, but if I wanted to consciously make a protein - say, as a therapeutic - how would I go about doing this?

It turns out it’s easier not to make the protein yourself but rather to ask someone else - or actually something else - to make the protein for you. We call these artificially manufactured molecules recombinant proteins. Cells from all forms of life have been repurposed for the production of recombinant proteins. The system you choose depends on what kind of protein you want to make.

I learnt to make recombinant proteins using E. coli - my PhD smelled of Luria Broth from autoclaving litres of the stuff, the food of choice for E. coli. LB is a nutrient-rich broth that smells something like nutritional yeast or vegetable stock powder - or perhaps even beef and onion crisps. Tasty. The proteins I made were lipid transfer proteins that picked up a specific type of lipid, moved it from one membrane to another, and exchanged it for a specific other lipid. Tiny protein machines whose specific job in all cells is to move specific lipids around.

I gave the instructions the E. coli needed to make the lipid transfer proteins in a small circle of DNA called a plasmid. E. coli works very well for some proteins, but there are limitations to the size of message it can read and the complexity of the protein it can make. This has led scientists to explore other systems.

Like many other people [1], during the pandemic my house plant collection ballooned - among others, we now have a row of carnivorous plants on our kitchen windowsill - pitcher plants, Monkey Cups, an escaped sundew, and of course the go-to Venus fly trap. These plants have evolved to grow on poor-nutrient soils by capturing and digesting insects and other small animals. The leaves of pitcher plants form little cups, strong enough to hold an enticing pool of liquid. But don’t be fooled by this - the purpose of this liquid is not the selfless hydration of animals - this liquid contains all manner of digestive enzymes that ensure the calculated decomposition and efficient absorption of the insect prey. (We’ll be meeting an enzyme that breaks down a particular kind of lipid again a bit later.)

So convenient are these little pools of proteins that some scientists have figured out how to engineer pitcher plants (Nepenthes mirabilis) to fill their pools with recombinant proteins. Plant Advanced Technologies (PAT) hold the patent for making recombinant protein in this way, and a subsidiary of PAT, Temisis Therapeutics, have a small molecule in pre-clinical trials for the treatment of psoriasis - although the detail of what that molecule is and how it is made is not revealed.

Using plants at scale to make recombinant proteins has many advantages over using animals or bacteria to perform the same role, not least their cost. Plants, like animal cells - but unlike bacteria - are able to make, fold and assemble complex proteins, and also to add embellishments that can improve the safety of the recombinant protein when used as a therapeutic.

Protalix Biotherapeutics was the first company to gain approval from the US Food and Drug Administration for a therapeutic made by plants. This was Elelyso®, made using their ProCellEx® system of tobacco plant cell lines and approved for the treatment of Gaucher Disease in May 2012.

Winding forward, Protalix’s latest recombinant protein therapeutic was approved in the EU and US last year for the treatment of adult patients with Fabry disease. This is a rare genetic disease resulting from a fault in the α-galactosidase A protein. I mentioned earlier that we’d meet another enzyme that breaks down lipids - the job of α-galactosidase A is to digest a lipid called a sphingolipid. The resultant malfunctioning enzyme means that this sphingolipid accumulates in blood vessels and other organs of the body, causing blood vessels to narrow, reducing blood flow and the supply of nutrients to the parts of the body where they are needed. This includes the tiny filtering structures in the kidney, the glomeruli. If these filters can’t work properly, the body can’t maintain a balance between the waste it wants to get rid of and the important molecules it needs to function. This can lead to kidney disease, which can quickly become critical.

The currently available treatments for Fabry disease seek to replace the defective enzyme. Elfabrio® (pegunigalsidase alfa-iwxj), made by Protalix using their ProCellEx® plant-based expression system, is a modified version of the α-galactosidase A enzyme. This modified version has the addition of polyethylene glycol (PEG) causing the therapeutic to hang around longer in the body after being injected, meaning less injections are needed. It also means that the therapeutic is kinder to the immune system - it angers it less. And what’s more, this PEG can only be added by plants, not by bacterial expression systems.

Nepenthes x ventrata 'Monkey Cups'

Reference

1. Statista Research Department (2023). Annual houseplant sales change post-pandemic in the UK 2021. Statista. Accessed September 26, 2024.

Further Reading

Garner, K. L. (2021) Principles of synthetic biology Essays Biochem. 65(5): 791–811.

Miguel, S., Nisse, E., Biteau, F., Rottloff, S., Mignard, B., Gontier, E., Hehn, A. and Bourgaud, F. (2019) Assessing Carnivorous Plants for the Production of Recombinant Proteins Front. Plant Sci. 10

Wallace EL, Goker-Alpan O, Wilcox WR, et al. (2024) Head-to-head trial of pegunigalsidase alfa versus agalsidase beta in patients with Fabry disease and deteriorating renal function: results from the 2-year randomised phase III BALANCE study J. Med. Genet. 61:520-530.