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

Abstract

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.

Design and Construction of Highly Stable, Protease-resistant Chimeric Avidins

“…The mutated avidins were produced using a baculovirus expression system (8). Non-glycosylated AVR4-b was produced using the E. coli expression system (19) 3 to study the influence of the carbohydrate chains on the properties of the protein (Table I). Avidin proteins were purified effectively by 2-iminobiotin-agarose affinity chromatography.…”

“…Fluorescent Biotin Dissociation Assay-The binding of labeled biotin to avidins was analyzed by a method based on the quenching of a biotin-coupled fluorescent probe ArcDia TM BF560 (ArcDia Ltd., Turku, Finland) caused by binding to avidin as described previously (19). The measurements were performed using a PerkinElmer Life Sciences LS55 luminometer with thermostated cuvette (25 or 50°C).…”

Novel Avidin-like Protein from a Root Nodule Symbiotic Bacterium, Bradyrhizobium japonicum

“…The recombinant proteins were isolated from bacterial cell extracts by affinity chromatography on 2-iminobiotin agarose column (27). Eluted proteins were analyzed by SDS-PAGE and subsequent Coomassie staining of the gels.…”

“…Escherichia coli BL-21(AI) cells (Invitrogen) were used for protein expression as described previously (27). The recombinant proteins were isolated from bacterial cell extracts by affinity chromatography on 2-iminobiotin agarose column (27).…”

“…The dissociation rate of a fluorescent biotin conjugate (ArcDia BF560 TM -biotin) was measured, as described previously (27), at 50°C. The purified proteins were analyzed by gel filtration using a Shimadzu HPLC instrument equipped with a Superdex 200 HR 10/30 column (Amersham Biosciences) with 50 mM sodium carbonate buffer (pH 11) with 150 mM NaCl as the liquid phase.…”