Therapeutic proteins play an important role in modern medicine. Correct glycosylation patterns are therefore fundamentally important for producing effective glycoprotein-based therapeutics. Dr. Marina Rubini from the University of Konstanz explores ways to alter the properties of proteins using non-natural amino acids for the post-translational site-specific attachment of carbohydrate groups. She uses the glycoprotein hormone erythropoietin (EPO), which is one of the most common prescription drugs worldwide, as a model.
Cancer patients often suffer not only from the cancer itself but also from the adverse effects of chemotherapy. Chemotherapeutic drugs act mainly by killing cells that divide rapidly, which is one of the main properties of cancer cells. However, as well as attacking cancer cells, the drugs also attack blood cells that produce erythrocytes. As a consequence, many patients suffer from anaemia, which is often treated with erythropoietin (EPO), a glycoprotein hormone that is critical in the formation of red blood cells. EPO is able to attenuate negative side effects of chemotherapies such as anaemia and improve prognosis.
In order to be able to supply sufficient quantities of EPO for therapeutic applications, it is produced with recombinant DNA technology in mammalian cell cultures. As EPO is a glycosylated protein, which means that carbohydrate chains (glycans) are attached to the hormone, it cannot be produced with bacteria. EPO therefore needs to be produced with mammalian cell cultures. “Bacteria cannot produce any glycosylated proteins,” explains Dr. Marina Rubini who works at the University of Konstanz and centres on rational protein design.
In mammalian cells, carbohydrate structures are attached to the proteins during or after translation. The type and pattern of the attached carbohydrates are crucial for the protein’s half-life and hence its biological activity. The carbohydrate chains also protect the protein against proteases which would otherwise degrade the protein within a relatively short time period.
The glycosylation pattern varies between different organisms as well as in individual cells, but there is no direct correlation between glycan structure and protein function. In order to improve this situation, Dr. Marina Rubini is developing a new approach for the production of stable protein variants with homogeneous glycosylation patterns by attaching specific glycans. “We use EPO as a model for the synthesis and optimisation of therapeutic proteins,” says Rubini explaining the background to her project.
The biochemist produces recombinant and stable EPO with a technique that enables her to integrate specific post-translational modifications in proteins as they are synthesised. Recombinant non-glycosylated EPO is produced in E. coli, rather than in mammalian cells. “Bacteria are perfect for producing recombinant proteins; they can be easily manipulated genetically and can also produce large quantities of protein,” Rubini says. In order to produce glycosylated proteins from the non-glycosylated ones, Rubini uses non-natural amino acids with specific functional side chains. Carbohydrate moieties are introduced into E. coli proteins by introducing a mutation at a desired site, resulting in a stop codon that cannot be translated into an amino acid. A stop codon within the messenger RNA normally signals the termination of translation. “In the system we use, this particular stop codon has a special function in that it serves as a signal for the incorporation of a non-natural amino acid,” said Dr. Rubini explaining the principle. The non-glycosylated protein is produced in bacteria, and the glycans, i.e. carbohydrate structures, are added later in the 'test tube'.
The carbohydrate moieties are conjugated with the amino acids using a technique known as click chemistry. Click chemistry is not a single specific reaction, but rather involves several reactions that generate substances by quickly and reliably joining small modular units. Dr. Rubini uses a reaction known as copper-catalysed cycloaddition. “The advantage of this approach is that high quantities of recombinant protein can be produced in E. coli; we can add virtually any type of modification later,” Dr. Rubini says.
In theory, the technology can also be used for the production of other therapeutic proteins. Dr. Rubini hopes that the use of specific carbohydrate chains will also provide her with new insights into the general interaction between glycans and proteins. “The new findings can then be used to increase the efficiency of EPO and, in the ideal case scenario, to optimise other therapeutic proteins,” says Dr. Rubini, explaining what she hopes to achieve. However, the early application of glycoproteins produced with Rubini’s new method is not likely. “Unfortunately, we are only just starting out and there is still a long way to go,” Dr. Rubini says seeking to dampen expectations.
Dr. Marina Rubini – between scientific curiosity and life-saving application
Marina Rubini did her degree thesis in Padua, Italy, and decided to stay in academic research. “My supervisor in Padua was responsible for my love for chemistry and also taught me to believe in myself,” recalls Dr. Rubini.
When she was doing her doctoral thesis at the Max Planck Institute for Biochemistry in Martinsried, Rubini came into contact with non-natural amino acids. “This was another decisive experience towards what continues to be my work today,” Rubini explains. At the University of Konstanz, she was finally able to fully develop her capabilities, first as post-doc and now as the head of a junior research group, where she has the freedom to choose her projects herself. Her decision to apply for a research position at the University of Konstanz was a completely spontaneous one, she says. After her first post-doctoral position, she thought about applying for a post in industry, but meeting Prof. Dr. Andreas Marx from the University of Konstanz at a scientific conference changed her mind. “I gave a talk, and Prof. Marx came up to me to ask whether I would be interested in working in his group in Konstanz and establishing the method used for incorporating non-natural amino acids in proteins. And I said yes straight away,” Rubini says.
Further information:Dr. Marina RubiniFaculty of ChemistryUniversity of KonstanzE-mail: Marina.Rubini(at)uni-konstanz.de