Research team improves method for producing designer proteins

A team led by protein chemist Prof Henning Mootz and PhD student Christoph Humberg from the Institute of Biochemistry at the University of Münster (Germany) has now answered a long-standing research question: Why does a special variant of the inteins, the 'split inteins', often encounter problems in the laboratory that significantly lower the efficiency of the reaction? The researchers were able to identify protein misfolding as one cause and have developed a method to prevent it.

The splicing of proteins rarely occurs in nature but is very interesting for research. The solution found by the Münster team opens up possibilities for using split inteins to produce proteins that are useful in basic research or for applications in biotechnology and biomedicine. Scientists around the world are working intensively on synthesising complex proteins from two fragments that are difficult or impossible to produce using conventional methods. This way, chimeric proteins can be obtained in which, for example, one part of the protein has been produced in mammalian cells, while the other part has been chemically synthesised, selectively modified or obtained from bacterial cells. For this purpose, particularly powerful split inteins are required as a tool. They can join separate protein parts together, as they consist of two fragments that are localised on the initially separated peptide chains. Once the parts are joined together, the split intein removes itself.

The researchers in Münster investigated the so-called 'Aes intein', which enables a particularly broad range of applications thanks to a rare form of catalysis. Both fragments of the split intein were produced in the laboratory in bacterial cells and demonstrated only low productivity, similar to that of other inteins. Using chromatographic and biophysical methods, the team discovered that a large proportion of one of the fragments produced was present as an inactive protein aggregate with a specific misfolding. From these findings, the researchers drew conclusions about the cause of the misfolding and used bioinformatic analyses to identify a few amino acids that are responsible for it. Using molecular biological methods, they introduced selected single mutations in the intein fragment, which almost completely suppressed the formation of the aggregates and increased the productivity of the split intein accordingly.