RAB Xpress

 

Establishment and application of a novel protein expression platform

Establishing and applying novel protein expression systems is crucial in producing the full repertoire of economically relevant proteins in the area of red and white biotechnology. Although a variety of prokaryotic and eukaryotic expression systems is already successfully applied in the industrial biotechnology, there is a great need for novel expression platforms that allow heterologous expression of proteins that are currently difficult to produce. To fill existing gaps, we use the basidiomycete fungus Ustilago maydis as novel cellular platform for protein production. This eukaryotic microorganism is very well suited for biotechnological applications as it is amenable to genetic manipulation and mutants can be generated rapidly. Furthermore, biochemical and proteomic approaches are well established and easily applicable.

 

Exploiting unconventional secretion for protein expression

Recent findings demonstrated that protein expression in this eukaryotic microorganism is extensively regulated at the posttranscriptional level. In particular, the key RNA-binding protein for long-distance mRNA transport, Rrm4, is essential for efficient unconventional secretion of the endochitinase Cts1 during filamentous growth. In our group, we aim to combine posttranscriptional regulation at the level of microtubule-dependent mRNA transport during filamentous growth with unconventional secretion to export proteins. We plan to use the Rrm4-dependent Cts1 secretion apparatus to export heterologous proteins of interest (Figure 1), i.e. difficult to express lipases or therapeutic antibody formats. We are going to develop and apply a custom-tailored system including, e.g. convenient expression vectors with RNA transport signals.

Figure 1. Endochitinase Cts1 (green) as a carrier for unconventional protein secretion. The process depends on mRNA-transport along polarized microtubules mediated by the RNA-binding protein Rrm4 (red). Click on image to enlarge

 

Our strategy

To achieve protein expression we follow an 8-step strategy (Figure 2): After codon-optimization of the nucleotide sequence of a product (step 1), we generate various expression vectors (step 2) followed by quick strain generation by using a novel locus for enhanced homologous recombination (step 3). Subsequently, the resulting mutants are screened for protein expression using Western blot analysis. Protein is then enriched or purified from supernatants and activity assays are performed to verify the biological activity (Steps 6 and 7).

Figure 2. 8-step strategy of our unconventional protein expression platform. Click on image to enlarge

 

At the same time we work on optimization of our expression system on different levels, e.g. by deletion of harmful proteases and modifying different components to eliminate bottlenecks (step 8). Optimized strains will be used to scale-up expression by establishing fermentation conditions for U. maydis. Thus, the unique combination of unconventional secretion and posttranscriptional control at the level of long-distance mRNA transport will result in a novel and efficient platform for protein expression.
As proofs-of-principle we already succeeded in expressing active ß-Glucuronidase (Gus) and a single-chain antibody directed against the c-Myc tag (scFv anti-cMyc, Figure 3) in our system. Different other proteins are currently in the pipeline.

Figure 3. Single-chain antibodies (scFv) against the c-Myc tag produced in U. maydis are functional. The endochitinase Cts1 deals as a carrier to target the protein to the supernatant using a novel unconventional secretory pathway. Click on image to enlarge

Kontakt

Institut für Mikrobiologie
Department Biologie
Heinrich-Heine-Universität
Düsseldorf
Universitätsstraße 1
Gebäude: 26.12
Etage: 01
40225 Düsseldorf
Tel.: +49 211 81-14720
Fax: +49 211 81-15370
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Molekulare Mykologie
Prof i. R. Dr. J. F. Ernst undefined

Verantwortlich für den Inhalt: E-Mail sendenProf. Dr. Michael Feldbrügge