5 Common Challenges in Protein Expression and How Plants Offer Solutions
Proteins are the building blocks of life, responsible for all sorts of vital functions in living organisms. They are also important for therapeutic development and as tools for scientific research where they are often made using recombinant methods. But making recombinant proteins isn't always that easy. There are several reasons why your proteins might not be expressing properly. A plant-based method of protein expression might just hold the solution to some common issues encountered with recombinant protein expression.
Let's explore the challenges and look at how plants can help overcome them.
1. Codon Usage and mRNA Structure
Proteins really are the building blocks of life, and they're made according to instructions from the DNA code which is transcribed into messenger RNA (mRNA). This mRNA is then translated into the amino acids that form the folded protein. Sounds simple right? Well not quite, there can often be some tricky bits along the way.
Depending on your choice of expression system, you will need to consider the preferred codon usage of the host cell. Just like a recipe needs the right ingredients, cells need the right blend of codons to help them make proteins most efficiently. Using the wrong codons can cause the ribosome (the cellular machine that turns mRNA into protein) to pause, which in turn can lead to premature termination of protein translation.
The secondary structure of the mRNA is similarly important because unstable mRNA can have a reduced half-life and a lower translation efficiency which will ultimately lead to a decrease in protein production. In recent years the use of synthetic genes that encode the target protein has meant we are no longer reliant on the codon usage and resultant mRNA structure provided by the native DNA.
At Leaf, we work with synthetic gene experts to optimise the DNA sequence to match the plant cell’s codon preferences, giving our plants the best possible starting point for protein expression; preventing translation hiccups and boosting protein production.
2. Incomplete Processing
Proteins are like pieces of a giant cellular puzzle, they need to be folded correctly into a specific shape in order to fit the specialised requirements of their roles. After they're translated, they might need a little help from chaperone proteins to fold correctly. In addition, some proteins even require extra processing to help them form stabilising disulphide bonds or chemical modifications like biotinylation, phosphorylation or glycosylation. These modifications often happen in specialised compartments “organelles” within the cells called endoplasmic reticulum or golgi apparatus.
Simple bacterial expression systems lack these organelles, and the enzymes that reside within them that are responsible for carrying out these processing functions. As a result, these prokaryotic expression systems can often struggle to produce many complex proteins in a correctly folded and functional form. In plants, these processing capabilities are built-in, thanks to their eukaryotic nature. It means they can handle complex proteins like monoclonal antibodies, with ease.
3. Protease Susceptibility
Proteases help to break down protein and support various processes, including degrading old proteins or aiding in protein maturation. When you express a foreign protein in a host cell it can sometimes trigger these proteases, leading to protein degradation. This can result in lower yields and the appearance of truncated forms of your protein which are unable to function correctly. Plants aren't immune to this issue, but at Leaf we've got a strategy to deal with this. By using signals encoded in the protein we can keep it safely within the endoplasmic reticulum, thereby reducing the risk of unwanted proteolysis and ensuring better protein accumulation. This can have the additional benefits of minimising the heterogeneity and complexity of glycosylation – a frequent challenge in the manufacture of protein-based therapeutics.
4. Intrinsic Instability
Proteins can form incredibly complex structures that are held together by a series of different molecular forces, including hydrogen bonds, dipole-dipole interactions, disulphide bridges and hydrophobic interactions. They’ve evolved to be stable within their natural environments. When you move them to a different cell type, you often lose the stabilising interactions provided by their native partners. This can lead to instability, misfolding, and low protein levels. Our plants grow in controlled environment rooms at 22°C. This low temperature helps to maintain the stability of the protein in the absence of its native partners. The plants also possess an abundance of specialised molecular chaperone proteins that can help fold and stabilize recombinant proteins.
5. Host Cell Toxicity
Cells in their native environment tightly regulate protein activity. But when you introduce a foreign protein it can disrupt the balance, leading to slower growth or even cell death. Alternatively, host cells might even recognise the new protein as foreign and target it for degradation or package it up in the inclusion bodies to prevent any unwanted effect within the cells. Plants offer a solution. Their cells are different from bacteria, insect or mammalian, making them more resilient to "toxic" proteins. They can also direct proteins to specific organelles to minimise harm to the host cell. This process can be manipulated by skilled protein scientists to enable accumulation of folded proteins in plant cells with minimal impact on plant growth. We have a panel of plant cell [expression] lines which are able to secrete the target protein into the growth media, allowing for high level accumulation whilst minimising the effects on the host cells.
In conclusion, the world of protein expression is full of challenges, but plants provide a unique platform to overcome many of these hurdles. With their built-in processing capabilities and adaptability, they're helping scientists and researchers produce proteins more effectively and efficiently. We are experts in producing proteins in plants, our SupraVec® technology allows us to deliver optimised genes to the plant cells which in turn has been fine-tuned to provide high levels of protein accumulation.