Peptides Containing Multiple Disulfide‐Bond Mosaic Expression in the Periplasm of Escherichia coli

Peptides containing multiple disulfide bonds are usually problematic when expressed in Escherichia coli. We conducted the expression of β-defensin with three disulfide bonds, hepcidin with four disulfide bonds, and DkTx with six disulfide bonds using a small leader protein mosaic expression in the periplasm of E. coli, and purified them by affinity chromatography and characterized them by mass spectroscopy. The result showed that the expression level was high. A large amount of the pure recombinant peptide was also recovered after purification with a Ni2+ affinity column. The mass results by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) indicated that the recombinant peptides had a folding structure, with the native status of all of the cysteines participating in disulfide bond formation. Moreover, after removing the tags, the result was identical to its natural form as well. We thus provide a method for producing large amounts of soluble peptides containing multiple disulfide bonds in E. coli.