Production of the biotechnologically relevant AFP from Aspergillus giganteus in the yeast Pichia pastoris.
The mould Aspergillus giganteus produces a basic, low molecular weight protein (AFP) showing in vitro and in vivo antifungal properties against important plant pathogens. AFP is secreted as an inactive precursor containing an amino-terminal extension of six amino acids (lf-AFP) which is later removed to produce the active protein. The molecular basis to explain this behavior and the features that determine the fungal specificity of this protein are not completely solved. In this work, the mature AFP (AFP *) and a version of AFP with an extended amino-terminal (proAFP) have been cloned and produced in the yeast Pichia pastoris. The two proteins have been purified to homogeneity and characterized from structural and functional points of view. Recombinant AFP * produced is practically indistinguishable from the natural fungal protein in terms of its spectroscopic and antifungal properties while proAFP is mostly inactive under identical assay conditions. The availability of an active AFP protein produced in P. pastoris will permit investigation of the mode of action and targeting specificity of AFP by using site-directed mutagenesis approaches.
López-García
Belén
B
Centre de Recerca en Agrigenómica (CRAG) CSIC-IRTA-UAB, Departamento de Genética Molecular, Instituto de Biología Molecular de Barcelona, CSIC, Jordi Girona 18, 08034 Barcelona, Spain.
Moreno
Ana Beatriz
AB
San Segundo
Blanca
B
De los Ríos
Vivian
V
Manning
James M
JM
Gavilanes
José G
JG
Martínez-del-Pozo
Alvaro
A
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000378:metabolism* / Q000494:pharmacology; D001230:Aspergillus / Q000235:genetics; D001483:Base Sequence; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D005670:Fusarium / Q000187:drug effects; D008969:Molecular Sequence Data; D010843:Pichia / Q000378:metabolism
D000935:Antifungal Agents; D005656:Fungal Proteins; C097609:antifungal protein, Aspergillus