Expression of a plant protein by Neurospora crassa.
Heterologous expression of plant genes may serve as an important alternative for producing plant proteins. We have investigated the ability of the fungus Neurospora crassa to secrete zeamatin, a protein produced by Zea mays. Zeamatin was induced after being fused to glucoamylase, an extracellular hydrolase produced by N. crassa. Glucoamylase induction and other culture parameters were monitored in untransformed N. crassa grown in shaken liquid culture. A DNA plasmid, pGEZ, was constructed by inserting zeamatin-encoding cDNA into an expression cassette containing the promoter, a truncated open reading frame, and the terminator sequence of the N. crassa glucoamylase gene. Zeamatin-encoding cDNA was modified at the N terminus to include a kex-2 protease site, allowing cleavage of the chimeric product in the secretory pathway. Strains containing the chimeric gene construct were grown in liquid culture and induced for glucoamylase and zeamatin production. Zeamatin antibody detected a protein in a Western blot of concentrated culture supernatants that comigrated with authentic zeamatin. Secreted zeamatin was active in inhibiting the growth of Candida albicans in an agar diffusion assay, indicating that zeamatin had been correctly synthesized, processed, and secreted by N. crassa.
Rasmussen-Wilson
S J
SJ
MycoTox, Inc., Denver, Colorado 80262, USA. swilso@mycotox.abti.com
Palas
J S
JS
Wolf
V J
VJ
Taft
C S
CS
Selitrennikoff
C P
CP
D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D002176:Candida albicans / Q000187:drug effects; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D014170:Transformation, Genetic; D014361:Trypsin Inhibitors; D003313:Zea mays / Q000235:genetics
D000935:Antifungal Agents; D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014361:Trypsin Inhibitors; C073468:zeamatin protein, Zea mays; D005087:Glucan 1,4-alpha-Glucosidase