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 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 5.8-7.5mg 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 1.8-2.8mg of purified recombinant protein per liter of culture. The recombinant peptides displayed functional properties similar to those of native BgK or BgK(F6A).
Département d'Ingénierie et d'Etudes des Protéines, CEA Saclay, 91191 Gif sur Yvette cedex, France.
Sandrine
S
Braud
Pascal
P
Belin
Janie
J
Dassa
Liliana
L
Pardo
Gilles
G
Mourier
Antony
A
Caruana
Birgit T
BT
Priest
Paula
P
Dulski
Maria L
ML
Garcia
André
A
Ménez
Jean-Claude
JC
Boulain
Sylvaine
S
Gasparini
D000818:Animals; D016466:CHO Cells; D003064:Cnidarian Venoms / Q000096:biosynthesis*; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism*; D004594:Electrophysiology; D004926:Escherichia coli / Q000378:metabolism*; D026902:Potassium Channel Blockers / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012615:Sea Anemones / Q000378:metabolism*
D003064:Cnidarian Venoms; D026902:Potassium Channel Blockers; D011994:Recombinant Proteins; C104455:toxin BgK