English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

Background  Small, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from  Neosartorya fischeri  and PAF from  Penicillium chrysogenum,  are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure–function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly  15 N-/ 13 C-isotope labelled protein. To meet these requirements, we developed a  P. chrysogenum -based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses.    Results  The APs PAF, PAF mutants and NFAP were expressed in a  P. chrysogenum  ∆ paf  mutant strain that served as perfect microbial expression factory. This strain lacks the  paf -gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong  paf  promoter, and the presence of a  paf -specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus  Penicillium digitatum , which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI–MS, ECD and NMR spectroscopy and growth inhibition assays.    Conclusion  This study demonstrates the implementation of a  paf  promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different  Penicillium  species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure–function analyses.    Electronic supplementary material  The online version of this article (doi:10.1186/s12934-016-0586-4) contains supplementary material, which is available to authorized users.

microbial cell factories

A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses