A truncated glucoamylase gene fusion for heterologous protein secretion from Aspergillus niger.
The secreted yield of hen egg-white lysozyme (HEWL) from the filamentous fungus Aspergillus niger was increased 10-20-fold by constructing a novel gene fusion. The cDNA sequence encoding mature HEWL was fused in frame to part of the native A. niger gene encoding glucoamylase (glaA), separated by a proteolytic cleavage site for in vivo processing. Using this construct, peak secreted HEWL yields of 1 g/l were obtained in A. niger shake flask cultures compared to about 50 mg/l when using an expression cassette lacking any glaA coding sequence. The portion of glaA used in the gene fusion encoded the first 498 amino acids of glucoamylase (G498) and comprised its secretion signal, the catalytic domain and most of the O-glycosylated linker region which, in the entire glucoamylase molecule, spatially separates and links the catalytic and starch-binding domains.
Jeenes
D J
DJ
A.F.R.C. Institute of Food Research, Norwich Research Park, Colney, UK.
Marczinke
B
B
MacKenzie
D A
DA
Archer
D B
DB
D000818:Animals; D001234:Aspergillus niger / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D005260:Female; D005800:Genes, Fungal; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D009113:Muramidase; D005087:Glucan 1,4-alpha-Glucosidase