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.

Background: SV40 DNA replication system is a very useful tool to understand the mechanism of replication, which is a tightly regulated process. Many environmental and cellular factors can induce cell cycle arrest or apoptosis by inhibiting DNA replication. In the course of our search for bioactive metabolites from the marine sponges, psammaplin A was found to have some anticancer properties, the possible mechanism of which was studied. Methods: Cell viability was determined by Cell Counting Kit-8 (CCK-8) to count living RAW264.7 cells by combining 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) and 1-methoxy-phenazine methosulfate (1-methoxy-PMS). The effect of psammaplin A on DNA replication was carried out in SV40 DNA replication system in vitro. The activities of topoisomerase I and polymerase α-primase were measured by the relaxation of superhelical plasmid DNA and the incorporation of [³H]dTTP to the template respectively. The ssDNA binding activity of RPA was assessed by Gel Mobility Shift Assay (GMSA). Results: We have found that psammaplin A delivers significant cytotoxic activity against the RAW264.7 cell line. It was also found that psammaplin A could substantially inhibit SV40 DNA replication in vitro, in which polymerase α-primase is one of its main targets. Conclusion: Taken together, we suggest that psammaplin A-induced cytotoxicity may correlate with its inhibition on DNA replication. Psammaplin A has the potential to be developed as an anticancer drug.