Protein labelling without the baggage

Imagine trying to find your belongings in an airport if your baggage tags are attached to every suitcase, not just yours. This is the problem facing biologists trying to tag parts of proteins – one of the most commonly studied biological molecules. Dr Laurent Caron explains how Cambridge Research Biochemicals is collaborating with academia to make protein labelling more precise

Credit: Prof Andrea Brand & Dr James Chell, Gurdon Institute, Cambridge

Biologists studying proteins using fluorescence or Nuclear Magnetic Resonance (NMR) face a challenge. They need to tag sections of proteins to make them easy to see. But common labelling strategies using commercial kits like fluorescein isothiocyanate (FITC) mark everything chemically active on the protein. It’s the biological equivalent of labelling every suitcase on a plane with your name and address. There is an alternative – using proteins with chemical tags built into the right location – but few of these are available.

A new project aims to tackle this problem. Cambridge Research Biochemicals (CRB) has teamed up with chemist Dr Steven Cobb and structural biologist Dr Ehmke Pohl from Durham University to create a toolkit of proteins with built-in tags. One day these will be available for researchers to buy off-the-shelf.

The first toolkits will be chemically-tagged chemokines – a large family of proteins with a critical role in autoimmune disease. Chemokines help direct the immune response and have an important role in HIV infection, cancer, asthma, allergies, and other diseases.

Creating these toolkits is a huge challenge. To succeed, the project will draw upon the skills of two academic disciplines and industry. The first step of the process – designing the chemical tags – needs a chemist. Cobb has created a small library of chemical tags already and can build more. The tags are chemically altered amino acids – the building blocks of proteins. Cobb will modify the amino acids so they show up clearly on NMR or fluoresce.

The smallest modification Cobb will make is swapping a hydrogen atom in an amino acid for an atom that shows up clearly on NMR such as Fluorine-19. The amino acid is typically phenylalanine, which is the simplest to modify without significantly changing its function. A larger modification is bolting a fluorescent molecule to other amino acids such as lysine.

Credit: Tamily A Weissman, Harvard University

After Cobb has designed a chemical tag, the next stage in the process is quickly and efficiently assembling the tagged and untagged amino acids into a protein chain. This is where CRB comes in. The company is the world’s second oldest manufacturer of peptides – polymers of amino acids linked by amide bonds. CRB can apply this experience to help them rapidly assemble proteins such as the chemokine CCL2, a 76 amino acid long peptide.

Last, but not least, Pohl will examine the modified proteins’ structure using crystallography to make sure they’re the correct shape. Proteins fold automatically into shapes that help them work correctly in cells. Chemically modifying natural amino acids could introduce a spanner into the works – preventing the protein from folding properly.

You might be asking why these toolkits haven’t been created before. An important reason is creating protein toolkits requires multidisciplinary collaboration and a willingness to work with industry. Cobb and the staff at CRB will learn which proteins and labels biologists need for their work from Pohl. We will use this to better meet our clients’ needs. Pohl’s skills in structural biology are crucial to testing the finished proteins.

Another reason why a toolkit hasn’t been created is chemically assembling proteins is traditionally low yield, skill-intensive and – until a few years ago – painfully slow. The first problem is amino acids have to be bolted one-by-one onto the growing protein chain. A critical parameter for successfully assembling a long peptide is the coupling efficiency. Incomplete reactions dramatically affect the purity of the peptide as the chain is elongated. Peptides longer than 50 amino acids still represent a challenge for peptide manufacturers.

Amino acids are added individually to the chain using the well-known technique of solid-phase peptide synthesis (SPPS). To make a peptide using SPPS, the first amino acid in the peptide chain is bonded at one end to a resin support. A mixture of amino acid and coupling reagent is washed over the resin. The resin must be cleaned of unwanted reagents before the next amino acid can be added. Once all the amino acids have been added, the peptide chains are detached from the support and purified.

“Common labelling strategies mark everything chemically active on the protein. It’s the biological equivalent of labelling every suitcase on a plane with your name and address”

The second problem assembling proteins is methods for peptide purification, such as high-performance liquid chromatography, work poorly beyond 30 amino acids. Purification is essential because of incomplete couplings during synthesis or side-reactions during cleavage result in a crude peptide, a mixture of peptides varying in length – some with the correct length, but others missing several amino acids.

CRB will get around this problem using a technique called ligation, which has become possible during the last couple of years. This involves creating two or three short peptide chains and binding the pieces together into the finished protein. Take the chemokine CCL2, which is 76 amino acids long. This could be created from three peptide fragments 26, 25 and 25 amino acids long.

Peptide synthesis has also speeded up during the last few years. A decade ago, synthesising a protein could take months. Each chemical reaction between the amino acids was done at room temperature. Unlike in chemistry where heat is used to accelerate reactions, biological molecules like proteins break down under heat.

Microwave energy is now used to gently warm and speed up reactions without damaging the amino acids. Both CRB and Durham University own a Liberty peptide synthesiser manufactured by CEM. A peptide can be synthesised in two hours, according to the manufacturer – a 10 to 20-fold increase on non-microwave assisted synthesis. Proteins can be manufactured in days.

Rapid peptide synthesis using microwave technology

But it’s not only the heating that has speeded up reaction times and improved the quality of synthesised peptides. Amino acid manufacturers have developed better reagents. They’ve also worked to reduce or remove unwanted chemical reactions between amino acids, reagents and resins that can pollute peptide products.

CRB, Cobb and Pohl will spend the next 18 months creating the first library of tags using chemokine CCL2. Once this is complete, we will move onto CCL1, CCL5 and other chemokines. We’ve selected chemokines because they are technically challenging to synthesise and crystallise, but not insurmountably difficult. Chemokines are also biologically important.

There are almost infinite possibilities for tags we could develop. Many applications exist for NMR and fluorescence microscopy techniques. Biologists use chemical tags for countless different experiments, including studying interactions between two or more proteins, and interactions between proteins and other molecules. CRB will determine which tags to develop using its long experience of meeting its customers’ needs.

One important application of chemical tags on chemokines is in drug development. Chemokine receptor antagonists are drugs with a huge potential market treating autoimmune diseases like rheumatoid arthritis and inflammatory bowel disease. In 2006, the estimated market for autoimmune diseases was more than US$21bn. Yet these drugs have routinely failed at clinical trial.

More chemical tags for chemokines could help drug developers observe and understand why many chemokine receptor antagonists failed to work. One reason these drugs are often ineffective is they interact weakly with chemokine proteins. They also often target one protein, but a disease may be linked to more than one chemokine. Improved chemical tags could help research and development teams see why binding is poor and develop drugs that bond to several proteins.

Developing chemical tags for non-chemokines may be several years away, but the sky is the limit. Proteins are critical to all cell processes and structures. Biologists need to know the exact location of a chemical tag for many experimental techniques. CRB and our academic partners will be guided by the needs of our customers as we develop new toolkits of chemically-tagged proteins in the years ahead.

The Author:

Dr Laurent Caron is Peptide Core Technology Manager at CRB and CRB's technical coordinator on the BIOSCENT project, a pan-European R&D Framework Plan seven project worth €6m where CRB is responsible for providing peptide synthesis expertise.