Avidin is a promising tag for fusion proteins produced in baculovirus-infected insect cells.
The baculovirus expression vector system (BEVS) has become one of the most versatile and powerful eukaryotic systems for recombinant protein expression. We have constructed a novel baculovirus transfer vector (pbacAVs+C) which allows for the efficient production, detection, and single-step purification of the desired molecule as a secretion-compatible avidin fusion protein in insect cells. It also enables fast construction of the baculoviruses by site-specific transposition in Escherichia coli. To demonstrate the power of this vector, we report here on the production of immunologically intact hevein, a major cysteine-rich latex allergen, as avidin fusion protein. Our results indicate that avidin is a stable and versatile tag in the BEVS. It retains its extraordinarily high biotin-binding activity and also enables independent folding of the fusion partner. The versatility with which avidin fusion proteins can be detected, purified, and immobilized is the basis for the use of our system as a useful alternative in eukaryotic fusion protein production.
Airenne
K J
KJ
Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylä, FIN-40100, Finland.
Laitinen
O H
OH
Alenius
H
H
Mikkola
J
J
Kalkkinen
N
N
Arif
S A
SA
Yeang
H Y
HY
Palosuo
T
T
Kulomaa
M S
MS
D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides; D001360:Avidin / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004765:Enteropeptidase; D015870:Gene Expression; D005822:Genetic Vectors; D037102:Lectins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D037121:Plant Lectins; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D018411:Spodoptera
D023181:Antimicrobial Cationic Peptides; D017931:DNA Primers; D037102:Lectins; D037121:Plant Lectins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C045875:hevein; D001360:Avidin; D004765:Enteropeptidase