BgK, a disulfide-containing sea anemone toxin blocking K + channels, can be produced in Escherichia coli cytoplasm as a functional tagged protein

BgK, a sea anemone peptide consisting of 37 amino acid residues and 3 disulfide bonds, blocks voltage-gated potassium (Kv1) channels. Here, we report a method for producing tagged BgK in Escherichia coli, as a soluble cytoplasmic protein. First, using peptidic synthesis, we show that addition of a 1...

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Bibliographic Details
Published in:Protein expression and purification 2004-11, Vol.38 (1), p.69-78
Main Authors: Braud, Sandrine, Belin, Pascal, Dassa, Janie, Pardo, Liliana, Mourier, Gilles, Caruana, Antony, Priest, Birgit T., Dulski, Paula, Garcia, Maria L., Ménez, André, Boulain, Jean-Claude, Gasparini, Sylvaine
Format: Article
Language:English
Subjects:
Animals
CHO Cells
Cnidarian Venoms - biosynthesis
Cricetinae
Cricetulus
Cytoplasm - metabolism
Cytoplasmic production
Disulfide
Electrophysiology
Escherichia coli - metabolism
Potassium channel
Potassium Channel Blockers - metabolism
Recombinant Proteins - biosynthesis
Recombinant Proteins - isolation & purification
Sea anemone toxin
Sea Anemones - metabolism
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Summary:BgK, a sea anemone peptide consisting of 37 amino acid residues and 3 disulfide bonds, blocks voltage-gated potassium (Kv1) channels. Here, we report a method for producing tagged BgK in Escherichia coli, as a soluble cytoplasmic protein. First, using peptidic synthesis, we show that addition of a 15 residue peptide (S·Tag) at the BgK C-terminus does not affect its biological activity. Then, a synthetic DNA sequence encoding BgK was constructed and cloned to produce a BgK-S·Tag hybrid in the cytoplasm of E. coli. The presence of S·Tag did not only facilitate detection, quantification, and purification of the recombinant protein, but also increased the production yield by more than two orders of magnitude. Moreover, use of an E. coli OrigamiB(DE3)pLacI strain also increased production; up to 5.8–7.5 mg of BgK-S·Tag or mutated BgK(F6A)-S·Tag was produced per liter of culture and could be functionally characterized in crude extracts. Using a two-step purification procedure (affinity chromatography and RP-HPLC), we obtained 1.8–2.8 mg of purified recombinant protein per liter of culture. The recombinant peptides displayed functional properties similar to those of native BgK or BgK(F6A).
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2004.07.011