Title
Glucoamylasegreen fluorescent protein fusions to monitor protein secretion in Aspergillus niger
Author
TNO Voeding
Gordon, C.L.
Khalaj, V.
Ram, A.F.J.
Archer, D.B.
Brookman, J.L.
Trinci, A.P.J.
Jeenes, D.J.
Doonan, J.H.
Wells, B.
Punt, P.J.
van den Hondel, C.A.M.J.J.
Robson, G.D.
Publication year
2000
Abstract
A glucoamylase::green fluorescent protein fusion (GLA::sGFP) was constructed which allows the green fluorescent protein to be used as an in vivo reporter of protein secretion in Aspergillus niger. Two secretory fusions were designed for secretion of GLA::sGFP which employed slightly different lengths of the glucoamylase protein (GLA499 and GLA514). Expression of GLA::sGFP revealed that fluorescence was localized in the hyphal cell walls and septa, and that fluorescence was most intense at hyphal apices. Extracellular GLA::sGFP was detectable by Western blotting only in the supernatant of young cultures grown in soya milk medium. In older cultures, acidification of the medium and induction of proteases were probably responsible for the loss of extracellular and cell wall fluorescence and the inability to detect GLA::sGFP by Western analysis. A strain containing the GLA::sCFP construct was subjected to UV mutagenesis and survivors screened for mutations in the general secretory pathway. Three mutants were isolated that were unable to form a halo on either starch or gelatin medium. All three mutants grew poorly compared to the parental strain. Fluorescence microscopy revealed that for two of the mutants, GLA::sGFP accumulated intracellularly with no evidence of wall fluorescence, whereas for the third mutant, wall fluorescence was observed with no evidence of intracellular accumulation. These results indicate that the GLA::sGFP fusion constructs can be used as convenient fluorescent markers to study the dynamics of protein secretion in vivo and as a tool in the isolation of mutants in the general secretory pathway. Chemicals / CAS glucan 1,4 alpha glucosidase, 9032-08-0
Subject
Aspergillus niger
Glucoamylase
Green fluorescent protein (GFP)
Heterologous protein production
Protein secretion
glucan 1,4 alpha glucosidase
green fluorescent protein
hybrid protein
cell wall
fluorescence
fluorescence microscopy
fungus culture
fungus mutant
mutagenesis
nonhuman
protein secretion
Western blotting
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DOI
https://doi.org/10.1099/00221287-146-2-415
TNO identifier
953526
ISSN
1350-0872
Source
Microbiology, 146 (146), 415-426
Document type
article