Efficient production of active chicken avidin using a bacterial signal peptide in Escherichia coli

A1 Originalartikel i en vetenskaplig tidskrift (referentgranskad)


Interna författare/redaktörer


Publikationens författare: Hytönen VR, Laitinen OH, Airenne TT, Kidron H, Meltola NJ, Porkka EJ, Hörhä J, Paldanius T, Määttä JAE, Nordlund HR, Johnson MS, Salminen TA, Airenne KJ, Ylä-Herttuala S, Kulomaa MS
Förläggare: PORTLAND PRESS LTD
Publiceringsår: 2004
Tidskrift: Biochemical Journal
Tidskriftsakronym: BIOCHEM J
Volym: 384
Nummer: 2
Artikelns första sida, sidnummer: 385
Artikelns sista sida, sidnummer: 390
Antal sidor: 6
ISSN: 0264-6021


Abstrakt

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 nonspecific 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.


Nyckelord

avidin-biotin technology, bacterial, Biotin, chicken avidin, expression system

Senast uppdaterad 2019-10-12 vid 04:10