A team from UCLouvain's Institute of Condensed Matter and Nanosciences has established a self-assembly method for proteins, which are complex molecules. This represents a giant leap in the fields of biotechnology and biomedicine.
There are a variety of methods for immobilising molecules at interfaces, thus making it possible to modify the surface properties of the materials (adhesion, colour, optical properties, durability, etc.). However, this is much more difficult for complex molecules, such as proteins, which are nevertheless essential in fields such as biomedicine and biotechnology. Indeed, it’s difficult to obtain a high quantity of immobilised proteins and stable layers without damaging them (or in scientific jargon ‘while preventing their denaturation’). Aurélien vander Straeten, Anna Bratek-Skicki, Alain M. Jonas, Charles-André Fustin and Christine Dupont-Gillain discovered a trick to overcome this difficulty: assembling proteins layer by layer by freeing their electric charge.
What is a protein? What is its purpose?
To understand this promising discovery, let's go back to the fundamentals: what is a protein? It’s a biomacromolecule, that is, a very large living molecule composed of amino acids. Proteins are essential to any living organism; every protein has its own, unique, specific sequence of amino acids. A protein can play different roles:
- transport (soluble proteins such as haemoglobin, which transports oxygen);
- structure (insoluble proteins such as myosin, which allows for muscle contraction);
- defending the body from external attack (antibodies);
- performing chemical or biochemical transformations (enzymes that, for example, degrade proteins into smaller pieces for the body to absorb).
Why immobiliser proteins at interfaces?
An interface or surface is a border area between materials. What’s the purpose of putting proteins in contact with certain surfaces?
- To make a diagnosis: a blood test makes it possible to identify circulating molecules called antigens, which tell us about pathology. To achieve this, a protein which recognises the antigen is used. For example, the antibody that our body usually produces is isolated and then deposited on a plastic box. On this surface, the antibody can recognise its usual target and create a bond with the antigen molecule, which will generate a signal. Thus the doctor can make a diagnosis.
- To develop biomaterials or materials for medical use (e.g. prostheses): take the example of a cell culture for the purpose of tissue regeneration. To make the cells proliferate in large numbers, they must be grown on a surface (a plastic box for example). To achieve this, the surface is covered with proteins that serve as signalling cells, which allows them to cling and proliferate.
- To carry out chemical transformations: enzymes are proteins capable of inducing chemical reactions in living organisms. They can induce the same reactions outside of these organisms, to create new materials, new molecules, etc., according to a chemistry approach that’s greener because it’s carried out under the typical mild conditions of living organisms. If the enzymes are immobilised on the inner surface of a reactor, the reaction can be done continuously, which is very effective.
How are proteins mobilised today?
Today, only non-green methods exist to immobilise proteins at interfaces:
- Immobilisation with reagents: reagents are rarely harmless to the environment, and tend to damage the three-dimensional structure of proteins.
- Adsorption: this is spontaneous accumulation on the surface of proteins. Two problems arise, however: the three-dimensional structure of the protein tends to disappear, denaturing the molecule, and quantities are often very low.
These immobilisation strategies therefore have the drawback of strongly denaturing the proteins and thus causing them to lose their bioactivity.
Towards greener chemistry
For three years, the UCLouvain team has been working on an alternative based on the self-assembly of enzymes. These enzymes represent a wonderful alternative to current chemistry which is often performed in high temperature reactors and in organic solvents. Indeed, enzymes can induce chemical reactions at 37 degrees in an aqueous medium, which is ideal for green chemistry, less polluting and less energy consuming (no more need to heat to 400° C in solvents harmful to the environment). Still required are a sufficient number of proteins, preventing their denaturation, and assembling them from A to Z using a ‘green’ process – not so simple. Two problems are inherent to protein constitution:
- Protein heterogeneity: every protein is different. It’s therefore difficult to find a universal method for all proteins.
- Heterogeneity of a particular protein: a protein is made up of a combination of hundreds, if not thousands, of amino acids, of which there are 20 different types.
Assuming that a protein is a pearl necklace of which most pearls (amino acids) differ from one another, the team decided to wrap this heterogeneous necklace in a macromolecule, which is itself a necklace with identical pearls. In other words, the idea is to standardise the proteins by inserting them into another homogeneous and electrically charged macromolecule called a polyelectrolyte in order to form protein-polyelectrolyte (PPC) complexes. PPCs are uniform, making it easier to manipulate proteins, and they also have other advantages:
- ‘Layer-by-layer’ assembly: this standardisation makes it possible to produce ‘molecular lasagne’ or ‘layer-by-layer’ assemblies: a layer of positive polyelectrolyte molecules is deposited on the surface, then a layer of negative particles (PPC), and so on. It is thus possible to control very precisely the quantity of proteins immobilised on the surface by varying the number of layers of this ‘molecular lasagne’.
- A medium that remains aqueous: this ‘packaging’ of molecules retains water. The medium remains well hydrated, which is favourable because the proteins avoid denaturisation and retain all their activity and biological function.
To what concrete purpose?
Aurélien vander Straeten, Christine Dupont and their collaborators thus developed protein packaging. They then identified the circumstances in which the method worked. The good news is that it is applicable to many proteins and therefore potentially universal. Currently, Mr vander Straeten is working on a concrete application: an antibacterial dressing that promotes healing through immobilisation of an antibacterial protein. This method can contribute to the production of antibacterial coatings for prostheses or other materials, and could be used to implement alternatives to oil-based plastic materials. For example, lignin, a macromolecule from wood, could be degraded enzymatically and reassembled as a plastic material. In short, this method is independent of the surface and can be used with any protein, making the field of experimentation vast and promising.
1PhD student at the UCLouvain Institute of Condensed Matter and Nanosciences (IMCN). PhD scholarship, FRIA (Fonds pour la Recherche dans l’Industrie et l’Agriculture). 2Postdoctoral research from Poland who worked at IMCN on a Marie Curie European scholarship. 3Professor, IMCN, UCLouvain, working at the scientific cross-roads of polymers, self-assembly and nanotechnologies. 4Professor, IMCN, UCLouvain, expert in polymers and macromolecular and supramolecular chemistry. 5Professor, IMCN and Faculty of Bioengineering, UCLouvain, specialist of biological interfaces.
A glance at Christine Dupont's bio
Christine Dupont holds a bachelor’s degree in bioengineering (1995) and a PhD in agronomy and bioengineering (2000) from the UCLouvain Faculty of Bioengineering. After a postdoctoral fellowship at the University of Manchester (United Kingdom), she obtained a mandate as a postdoctoral researcher and then research associate (2005) of the National Fund for Scientific Research. She is currently full professor at UCLouvain, where she teaches courses in the fields of chemistry, nanobiotechnologies and biomaterials, and leads an active research team in the field of biointerfaces and the mastery of interactions between living and non-living materials.
A glance at Aurélien vander Straeten's bio
In 2010, Aurélien vander Straeten began his studies in bioengineering at UCLouvain. In 2013, he chose to specialise in chemistry and bioindustries and, one year later, embarked on the nanobiotechnologies, materials and catalysis option. During his master's degree, he carried out a month of research in oncology at the University of Maastricht, six months of study at KU Leuven, and a dissertation on vaccine formulations. In 2015, he graduated as a bioengineer and won a FRIA scholarship to finance his PhD in protein immobilisation at interfaces. He is in the final year of his PhD.