A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses.
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 uniformly15N-/13C-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.


CONCLUSIONS
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.
Division of Molecular Biology, Biocenter, Medical University of Innsbruck, Innrain 80-82, Innsbruck, 6020, Austria.
Christoph
C
Sonderegger
Division of Molecular Biology, Biocenter, Medical University of Innsbruck, Innrain 80-82, Innsbruck, 6020, Austria.
László
L
Galgóczy
Biotechnology Department, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Avenida Agustín Escardino 7, 46980, Paterna, Valencia, Spain.
Sandra
S
Garrigues
Department of Organic Chemistry, Faculty of Science and Technology, University of Debrecen, Egyetem tér 1, 4032, Debrecen, Hungary.
Ádám
Á
Fizil
Institute of Biochemistry, Biological Research Centre of Hungarian Academy of Sciences, Temesvári krt. 62, 6726, Szeged, Hungary.
Attila
A
Borics
Biotechnology Department, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Avenida Agustín Escardino 7, 46980, Paterna, Valencia, Spain.
Paloma
P
Manzanares
Division of Molecular Biology, Biocenter, Medical University of Innsbruck, Innrain 80-82, Innsbruck, 6020, Austria.|Sandoz GmbH, Biochemiestrasse 10, 6250, Kundl, Austria.
Nikoletta
N
Hegedüs
Division of Molecular Biology, Biocenter, Medical University of Innsbruck, Innrain 80-82, Innsbruck, 6020, Austria.
Anna
A
Huber
Biotechnology Department, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Avenida Agustín Escardino 7, 46980, Paterna, Valencia, Spain.
Jose F
JF
Marcos
Department of Organic Chemistry, Faculty of Science and Technology, University of Debrecen, Egyetem tér 1, 4032, Debrecen, Hungary.
Gyula
G
Batta
Division of Molecular Biology, Biocenter, Medical University of Innsbruck, Innrain 80-82, Innsbruck, 6020, Austria. florentine.marx@i-med.ac.at.
Florentine
http://orcid.org/0000-0002-8408-1842
F
Marx
D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000737:chemistry; D002942:Circular Dichroism / Q000379:methods; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D016297:Mutagenesis, Site-Directed; D010408:Penicillium chrysogenum / Q000235:genetics / Q000378:metabolism*
D023181:Antimicrobial Cationic Peptides; D005656:Fungal Proteins; C097832:PAF protein, Penicillium chrysogenum; D003545:Cysteine
Antifungal proteins; Electronic circular dichroism (ECD) spectroscopy; NFAP; Neosartorya fischeri; Nuclear magnetic resonance (NMR); PAF; Penicillium chrysogenum; Penicillium digitatum; Recombinant protein production