Biotechnology has become an integral part of modern industry, especially in the realm of heterologous protein production. Various organisms serve as biotechnological production platforms, and among them, the fungus Ustilago maydis has gained increasing[K1] interest. Recent research has uncovered an unconventional secretion pathway that proves advantageous for exporting distinct heterologous proteins, with the chitinase Cts1 acting as a carrier. The system seems to be especially beneficial for exporting large proteins; however, the exact size limits are unknown.

This novel system has already been used to produce antibody formats like nanobodies. Nanobodies are emerging as novel and valuable products in modern industry due to their simplified structure compared to regular immunoglobulin antibodies. Simple production and the ability to easily multimerize them into large homo- or heterovalent fusion proteins offer significant advantage. The latter for example allows for the simultaneous detection of multiple targets or an increase in affinity against specific targets.

To determine the size limits of heterologous unconventional secretion cargo, we here focus on multimerization studies using nanobodies against fluorescent proteins as a proof-of-principle with an easy read-out.

As a prerequisite, production and unconventional secretion of novel nanobodies targeting mCherry and Gfp could successfully be established. In the next step, we successfully generated functional heterobivalent dimers. Now, we plan to further expand the study and multimerize the nanobodies further.