Recombinant core-streptavidin

Details Recombinant core-streptavidin Recombinant core-streptavidin

1994-04-28

1996-02-06

Wax, Robert A.

Chemistry: molecular biology and microbiology

Spore forming or isolating process

536 237, 4352523, 4353201, C12N 121, C12N 510, C12N 1531, C12N 1563

Patent

active

054895286

DESCRIPTION:

BRIEF SUMMARY
Streptavidin in its natural form is a tetrameric protein, each subunit being 159 amino acids in length and having a molecular weight of 16,450 Da. Streptavidin is obtained from the culture filtrate of Streptomyces avidinii (Chaiet, L. et al., Antimicrob. Agents Chemother. 3 (1963), 28-32). A characteristic of streptavidin, as also of the homologous protein avidin isolated from chicken egg-white, is an exceptionally strong non-covalent binding to the water-soluble vitamin H (d-biotin). One molecule of biotin binds per streptavidin subunit, i.e. four molecules of biotin bind to the tetramer. The dissociation constant of this binding is 10.sup.15 mol/l. In contrast to avidin, the streptavidin derived from Streptomyces avidinii is not glycosylated, does not contain any amino acids containing sulphur and has a lower isoelectric point at ca. pH 6.5. The avidin-biotin system and in particular the streptavidin-biotin system is already widely used in diagnostics and molecular biology (Wilchek, M. and Bayer, E. A., Methods Enzymol. 184 (1990), 5-13 and 14-45). An example of the use of the biotin-streptavidin system is to attach biotin or streptavidin to target molecules, e.g. analytes or to a surface such as e.g. a reagent vessel surface, chromatographic media or biopolymers thus enabling the target molecule to be immobilized or detected.
Streptavidin is usually isolated as a secreted protein from the culture filtrate of Streptomyces avidinii. However, these streptavidin preparations are heterogeneous with regard to the N-terminal and/or C-terminal amino acid sequence which is due to proteolysis during the fermentation (long fermentation periods) or/and the purification (Argarana, C. E. et al., Nucl. Acids Res. 14 (1986) 1871-1882; Bayer, E. A. et al., Methods Enzymol. 184 (1990), 80-89; Pahler, A. et al., J. Biol. Chem. 262 (1987) 13933-13937; Hendrickson, W. A. et al., Proc. Natl. Acad. Sci. 86 (1989) 2190-2194; Bayer, E. A. et al., Biochem. J. 259 (1989) 369-376). In addition the complete native streptavidin tends to aggregate (Pahler, A. et al., J. Biol. Chem. 262 (1987) 13933-13937; Bayer, E. A. et al., Biochem. J. 259 (1989) 369-376; Bayer, E. A. et al., J. Biochem. Biophys. Methods 13 (1986) 103-112). As a consequence it is difficult to reproducibly isolate native or biologically active, proteolytically-shortened streptavidin proteins from S. avidinii.
The aggregation of native streptavidin to form oligomers which already occurs during the isolation and its low solubility (Pahler, A. et al., J. Biol. Chem. 262 (1987), 13933-13937) also leads to problems when streptavidin is used in a streptavidin-biotin system since a not exactly determinable portion of the streptavidin is withheld from the reaction mixture so that falsifications of the measured results are possible.
A further disadvantage of native streptavidin is that during fermentation and preparation it is proteolytically processed to a limited extent at its N and C terminus. Thus one usually obtains a mixture of different degradation products and the final product of this proteolytic processing is a "core" protein with about 125 to 127 amino acids (Pahler, A. et al., J. Biol. Chem. 262 (1987) 13933-13937; Bayer, E. A. et al., Biochem. J. 259 (1989) 369-376). The proteolytic processing improves the binding properties of streptavidin to biotin conjugates (Bayer, E. A. et al., Biochem. J. 259 (1989) 369-376). However, the mixture of degradation products that is usually obtained does not have exactly reproducible binding properties so that falsifications of the measured results are possible.
The recombinant production of streptavidin is described in EP-B 0 198 015. The heterologous expression and secretion of native streptavidin in E. coli by means of the native streptavidin signal sequence resulted in streptavidin variants which are secreted into the periplasma. However, these streptavidin variants denoted ECO avidins are also heterogeneous at their C terminus. Moreover the S. avidinii signal sequence is not completely cleaved in E. coli and as a result th

REFERENCES:
patent: 4839293 (1989-06-01), Cantor et al.
patent: 5328985 (1994-07-01), Sano et al.
Sano et al., Biochem. Biophys. Res. Comm. 176:571-577 (Apr. 30, 1991).
Sano et al., Proc. Natl. Acad. Sci. USA 87:142-146 (1990).
Bayer et al., Meth. Enzymol. 184:80-89 (1990).
Agarana et al., Nucl. Acids Res. 14:1871-1882 (1986).
Pahler et al., J. Biol. Chem. 262: 13933-13937 (1987).
Meyhack et al., In Genetics & Molecular Biology of Industrial Microorganisms, Hershberger et al. (eds), Amer. Soc. Microbiol., Washington, DC, 1989, pp. 311-321.

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