Bacteria can produce recombinant proteins quickly and cost effectively. However, their physiological properties limit their use for the production of proteins in their native form, especially polypeptides that are subjected to major post-translational modifications. Proteins that rely on disulfide bridges for their stability are difficult to produce in Escherichia coli. The bacterium offers the least costly, simplest, and fastest method for protein production. However, it is difficult to produce proteins with a very large size. Saccharomyces cerevisiae and Pichia pastoris are the most commonly used yeast species for protein production. At a low expense, yeasts can offer high protein yields, generate proteins with a molecular weight greater than 50 kDa, extract signal sequences, and glycosylate proteins. Both eukaryotic and prokaryotic species maintain reducing conditions in the cytoplasm. Hence, the formation of disulfide bonds is inhibited. These bonds are formed in eukaryotic cells during the export cycle, under the oxidizing conditions of the endoplasmic reticulum. Bacteria do not have an advanced subcellular space, but in the oxidizing periplasm, they exhibit both export systems and enzymatic activities directed at the formation and quality of disulfide bonds. Here, we discuss current techniques used to target eukaryotic and prokaryotic species for the generation of correctly folded proteins with disulfide bonds.

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.

Hepcidin, an antimicrobial peptide produced by the liver, also controls the iron balance and regeneration in vertebrates. Two types of hepcidin (Hamp1 and Hamp2) have been found in the bodies of black rockfish (Sebastes schlegelii). The full-length cDNA of hepcidin was cloned to enable a study of the antibacterial roles of these two hepcidins (Hamp) in black rockfish. The antimicrobial function of recombinant hepcidins was tested both in vitro and in vivo by the synthesis in Escherichia coli of recombinant hepcidin (approximately 11 kDa) from black rockfish. The recombinant hepcidins inhibited the growth of two bacterial species, Streptococcus iniae FP5228 and Pseudomonas aeruginosa, at various concentrations, in vitro after 6 h post-incubation, respectively. During infection, the production of ferroportin was reduced, suggesting the preservation of iron to prevent microbial proliferation. In vivo administration of Hamp1, but not Hamp2, synthetic peptides induced a substantial reduction in the expression of ferroportin, suggesting that in black rockfish with two forms of hepcidin, ferroportin production is regulated by the iron-regulator Hamp1, and not by the dedicated antimicrobial Hamp2. The findings of this study suggest the various antimicrobial roles of these two types of hepcidin.

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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.