Unraveling the Mystery of Inefficient Split Inteins in Protein Splicing

Proteins are fundamental building blocks of life, composed of folded peptide chains formed by sequences of amino acids. They perform a wide range of functions, from maintaining cellular structures to catalyzing biochemical reactions. This functional diversity is further expanded by post-synthetic modifications to the peptide chains.

One such modification is protein splicing, a process in which an internal protein segment known as an intein excises itself from the peptide chain, enabling proper folding and functionality of the mature protein.

A research team led by protein chemist Professor Henning Mootz and PhD student Christoph Humberg at the Institute of Biochemistry, University of Münster, has tackled a longstanding question in this field: Why do certain intein variants—specifically split inteins—often show reduced reaction efficiency in laboratory experiments?

The team identified protein misfolding as a key contributing factor and developed a strategy to overcome it.

Although protein splicing occurs rarely in nature, it is of great interest to researchers. The approach developed by the Münster group opens new possibilities for using split inteins to construct proteins with value in both basic research and applied fields like biotechnology and biomedicine.

Globally, scientists are working to synthesize complex proteins from two separate fragments that are difficult—or even impossible—to produce by conventional means. This strategy enables the creation of chimeric proteins, in which one segment may be expressed in mammalian cells, while the other is chemically synthesized, selectively modified, or produced in bacterial systems.

To enable this, split inteins serve as essential molecular tools. They can join two initially separate peptide fragments, and once the linkage is complete, the intein removes itself from the final product.

The Münster researchers centered their work on the Aes intein, which is notable for its broad range of applications due to a unique catalytic mechanism. However, when both fragments of the Aes split intein were expressed in bacterial cells, they showed low productivity—similar to other intein systems.

Using chromatographic and biophysical methods, the team discovered that one of the fragments frequently formed inactive protein aggregates due to misfolding.

From this, they hypothesized the underlying causes and used bioinformatic tools to identify specific amino acids responsible for the misfolding.

By applying molecular biology techniques, they introduced precise single-point mutations into the intein fragment. These mutations significantly reduced aggregate formation and greatly improved the productivity of the split intein.