Products/Services Used | Details | Operation |
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PCR Cloning and Subcloning> | The optimized gene was chemically synthesized (Genscript, Piscataway, NJ) with restriction sites for NdeI at the 5′-end and XhoI at the 3′-end, for subcloning in pET28a (Novagen, Gibbstown, USA) in frame with a N-terminal His-Tag...The proteolytic activity of purified recombinant shewasin D was determined with several fluorogenic protease substrates: [(7-methoxycoumarin-4-yl)acetyl(MCA)-Lys]-Lys-Pro-Ala-Glu-Phe-Phe-Ala-Leu-[Lys-2,4-Dinitrophenyl (DNP)] (GenScript, Piscataway, USA), [MCA-Lys]-Leu-His-Pro-Glu-Val-Leu-Phe-Val-Leu-Glu-[Lys-DNP] (Genosphere Biotechnologies), [MCA-Lys]-Ala-Leu-Ile-Pro-Ser-Tyr-Lys-Trp-Ser-[Lys-DNP] (GenScript, Piscataway, USA)...Fractions that were immunostained with the anti-shewasin D antibody (GenScript, Piscataway, USA) were pooled, diluted and applied to a Mono Q 5/50 (GE Healthcare Life Sciences) connected to an FPLC system (DuoFlow-BioRad, Hercules, USA). | Get A Quote |
The widespread presence of pepsin-like enzymes in eukaryotes together with their relevance in the control of multiple biological processes is reflected in the large number of studies published so far for this family of enzymes. By contrast, pepsin homologs from bacteria have only recently started to be characterized. The work with recombinant shewasin A from Shewanella amazonensis provided the first documentation of this activity in prokaryotes. Here we extend our studies to shewasin D, the pepsin homolog from Shewanella denitrificans, to gain further insight into this group of bacterial peptidases that likely represent ancestral versions of modern eukaryotic pepsin-like enzymes. We demonstrate that the e... More