Determinants of the fidelity of processing glucoamylase-lysozyme fusions by Aspergillus niger.
Fusion proteins are used to enhance the yields of heterologous proteins secreted from filamentous fungi. In Aspergillus niger, the target protein is normally fused downstream of the carrier protein glucoamylase with a Lys-Arg KEX2-like cleavage site at the junction. This is cleaved in vivo to release mature protein but the processing is not always accurate. We have used N-terminal mutant lysozymes to vary the sequence immediately downstream of the KEX site, and also varied the amino acid sequence upstream of the KEX processing site, to study the fidelity of processing. The sequences both upstream and downstream of the KEX2 site affected the fidelity of cleavage. With some constructs, a range of processing sites were apparent and the relative proportions were time dependent in batch cultures of A. niger. Aberrant processing was related to the secondary-structure preferences of the amino acids in and around the KEX site. Downstream of the processing site, the fidelity of processing decreased in proportion to the tendency for helix formation.
Spencer
J A
JA
Institute of Food Research, Norwich Research Park, Colney, UK.
Jeenes
D J
DJ
MacKenzie
D A
DA
Haynie
D T
DT
Archer
D B
DB
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology*; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004795:Enzyme Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009113:Muramidase / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D009113:Muramidase; D005087:Glucan 1,4-alpha-Glucosidase