Expression, purification, and characterization of the recombinant calcium-binding equine lysozyme secreted by the filamentous fungus Aspergillus niger: comparisons with the production of hen and human lysozymes.
Equine lysozyme (EqL) has been expressed from a synthetic gene and secreted from a heterologous host, the filamentous fungus Aspergillus niger. By including 100 mM Ca2+ in the growth medium, secreted yields of more than 50 mg/liter could be achieved using polyvinylpyrrolidone (PVP) complete medium. In a soya medium yields of up to 150 mg/liter were achieved. The production of recombinant human lysozyme (HuL) from A. niger with yields of over 40 mg/liter was also achieved using PVP medium. Addition of Ca2+ to the growth medium reduced the yield of both HuL and hen egg white lysozyme (HEWL). Sequence differences between the three lysozymes, EqL, HuL, and HEWL, resulted in different susceptibilities to cleavage by A. niger proteases. An improved procedure for the purification of EqL and HuL from A. niger allowed separation of the proteins from pigments produced by the fungus. Detailed spectroscopic analysis, including 2D 1H NMR, for recombinant EqL and recombinant HuL confirm that both proteins possess their native structure and are purified to homogeneity.
Spencer
A
A
Institute of Food Research, Norwich Research Park, Colney Lane, Norwich, NR4 7UA, United Kingdom.
Morozov-Roche
L A
LA
Noppe
W
W
MacKenzie
D A
DA
Jeenes
D J
DJ
Joniau
M
M
Dobson
C M
CM
Archer
D B
DB
D000595:Amino Acid Sequence; D000818:Animals; D001234:Aspergillus niger / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D004247:DNA / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006736:Horses; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013045:Species Specificity
D011993:Recombinant Fusion Proteins; D004247:DNA; D009113:Muramidase; D002118:Calcium