Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli.
Chicken avidin is a highly popular tool with countless applications in the life sciences. In the present study, an efficient method for producing avidin protein in the periplasmic space of Escherichia coli in the active form is described. Avidin was produced by replacing the native signal sequence of the protein with a bacterial OmpA secretion signal. The yield after a single 2-iminobiotin-agarose affinity purification step was approx. 10 mg/l of virtually pure avidin. Purified avidin had 3.7 free biotin-binding sites per tetramer and showed the same biotin-binding affinity and thermal stability as egg-white avidin. Avidin crystallized under various conditions, which will enable X-ray crystallographic studies. Avidin produced in E. coli lacks the carbohydrate chains of chicken avidin and the absence of glycosylation should decrease the non-specific binding that avidin exhibits towards many materials [Rosebrough and Hartley (1996) J. Nucl. Med. 37, 1380-1384]. The present method provides a feasible and inexpensive alternative for the production of recombinant avidin, avidin mutants and avidin fusion proteins for novel avidin-biotin technology applications.
Hytönen
Vesa P
VP
Department of Biological and Environmental Science, NanoScience Center, P.O. Box 35 (YAB), FIN-40014 University of Jyväskylä, Finland.
Laitinen
Olli H
OH
Airenne
Tomi T
TT
Kidron
Heidi
H
Meltola
Niko J
NJ
Porkka
Eevaleena J
EJ
Hörhä
Jarno
J
Paldanius
Tiina
T
Määttä
Juha A E
JA
Nordlund
Henri R
HR
Johnson
Mark S
MS
Salminen
Tiina A
TA
Airenne
Kari J
KJ
Ylä-Herttuala
Seppo
S
Kulomaa
Markku S
MS
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D030161:Avian Proteins / Q000096:biosynthesis / Q000737:chemistry; D001360:Avidin / Q000096:biosynthesis* / Q000737:chemistry; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry; D001426:Bacterial Proteins / Q000096:biosynthesis*; D002645:Chickens / Q000235:genetics*; D048168:Escherichia coli K12 / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021382:Protein Sorting Signals / Q000235:genetics*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D030161:Avian Proteins; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D001360:Avidin; C078167:OMPA outer membrane proteins