Efficient heterologous expression and secretion in Aspergillus oryzae of a llama variable heavy-chain antibody fragment V(HH) against EGFR.
We have constructed a filamentous fungus Aspergillus oryzae that secretes a llama variable heavy-chain antibody fragment (V(HH)) that binds specifically to epidermal growth factor receptor (EGFR) in a culture medium. A major improvement in yield was achieved by fusing the V(HH) with a Taka-amylase A signal sequence (sTAA) and a segment of 28 amino acids from the N-terminal region of Rhizopus oryzae lipase (N28). The yields of secreted, immunologically active anti-EGFR V(HH) reached 73.8 mg/1 in a Sakaguchi flask. The V(HH) fragments were released from the sTAA or N28 proteins by an indigenous A. oryzae protease during cultivation. The purified recombinant V(HH) fragment was specifically recognized and could bind to the EGFR with a high affinity.
Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
Fumiyoshi
F
Okazaki
Jun-ichi
J
Aoki
Soichiro
S
Tabuchi
Tsutomu
T
Tanaka
Chiaki
C
Ogino
Akihiko
A
Kondo
D000818:Animals; D001236:Aspergillus oryzae / Q000235:genetics*; D002161:Camelids, New World / Q000235:genetics / Q000276:immunology*; D066246:ErbB Receptors / Q000276:immunology*; D008049:Lipase / Q000235:genetics; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D012233:Rhizopus / Q000201:enzymology / Q000235:genetics; D061905:Single-Domain Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies; D066246:ErbB Receptors; D008049:Lipase