Recombinant expression in Escherichia coli allows the simple, economical, and effective production of bioactive peptides. On the other hand, the production of native peptides, particularly those rich in disulfide bonds, is a major problem. Previous studies have reported that the use of carrier proteins for fusion expression can result in good peptide yields, but few are folded correctly. In this study, two transmembrane small proteins in E. coli, YoaJ and YkgR, which both orientate with their N-termini in cytoplasm and their C-termini in periplasm, were used for fusion expression. The recombinant production of two peptides, asteropsin A (ASPA) and β-defensin (BD), was induced in the periplasm of E. coli using a selected carrier protein. Both peptides were expressed at high levels, at yields of approximately 5-10 mg/L of culture. Mass spectrometry showed that the resulting peptide had the same molecular weight as their natural forms. After purification, single peaks were observed by reversed phase high-performance liquid chromatography (RP-HPLC), demonstrating the absence of isoforms. Furthermore, cytoplasmically expressed fusion proteins with a carrier at their C-termini did not contain disulfide bonds. This study provides new carrier proteins for fusion expression of disulfide bond-rich peptides in E. coli.
Eukaryotic expression systems are used widely and have the advantages of protein processing, proteolytic cleavage, disulfide bond formation, and posttranslational modification in contrast to the prokaryotic expression system. In the present study, peptide gene (olive flounder beta‐defensin or hepcidin) was inserted into the vector of pPIC9K, which involved the secretion signal and promoter AOX1. The colonies with high copy numbers of the target gene for high‐level expression were selected by G418. Approximately 30 mg/L for beta‐defensin and 25 mg/L for hepcidin was obtained from the culture medium supernatant. An ammonium sulfate salting‐out method was used for purification; this one‐step purification simplified the procedures, and the purification effect was good in terms of the purity and yield. The proteins from yeast itself could be isolated easily using the ammonium sulfate salting‐out method.
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 Ni 2+ 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.
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