Heterologous gene expression in Aspergillus niger: a glucoamylase-porcine pancreatic prophospholipase A2 fusion protein is secreted and processed to yield mature enzyme.
The cDNA gene encoding porcine pancreatic prophospholipase A2 (proPLA2) was cloned into an Aspergillus niger expression vector downstream of the glucoamylase (glaA) gene promoter region. When this construct was transformed into A. niger, no detectable PLA2 was produced. Evidence was obtained showing that the PLA2 gene was transcribed and that PLA2 is extremely susceptible to both intracellular and extracellular proteases of A. niger, thus indicating that translation products would be rapidly degraded. By fusing the proPLA2-encoding sequence to the entire glaA gene, secreted yields of PLA2 up to 10 micrograms/ml were obtained from a transformed protease-deficient strain of A. niger. PLA2 was secreted in young cultures as a fusion protein, but in older cultures, it was processed from the glucoamylase carrier protein. Secreted PLA2 was shown to be enzymatically active and to have the correct N-terminal amino acid (aa) sequence, although another form of processed PLA2 was also produced. This form included two aa of the proregion from PLA2. The potential for improving yields of secreted heterologous proteins from A. niger still further is discussed.
Roberts
I N
IN
AFRC Institute of Food Research, Norwich Laboratory, UK.
Jeenes
D J
DJ
MacKenzie
D A
DA
Wilkinson
A P
AP
Sumner
I G
IG
Archer
D B
DB
D000595:Amino Acid Sequence; D000818:Animals; D001234:Aspergillus niger / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D005822:Genetic Vectors; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D010741:Phospholipases A / Q000235:genetics* / Q000378:metabolism; D054467:Phospholipases A2; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine; D014170:Transformation, Genetic
D004277:DNA, Single-Stranded; D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D010741:Phospholipases A; D054467:Phospholipases A2; D005087:Glucan 1,4-alpha-Glucosidase