2018 ; Vol.28-12: 2079~2094
|Author||David Quintero, Jamie Carrafa, Lena Vincent, Hee Jong Lee, James Wohlschlegel, David Bermudes|
|Place of duty||California State University, Northridge,University of California at Los Angeles|
|Title||Co-expression of a Chimeric Protease Inhibitor Secreted Salmonella Protects Therapeutic Proteins from Proteolytic Degradation.|
J. Microbiol. Biotechnol.2018 ;
|Abstract||Sunflower trypsin inhibitor (SFTI) is a 14 amino acid peptide that contains a single internal disulfide bond. We initially constructed chimeras of SFTI with N-terminal secretion signals from the Escherichia coli and the Pseudomonas aeruginosa, but only detected small amounts of protease inhibition resulting from these constructs. A substantially higher degree of protease inhibition was detected from a C-terminal SFTI fusion with E. coli YebF, which radiated more than a centimeter from a single colony of E. coli using a culture-based inhibitor assay. Inhibitory activity was further improved in YebF-SFTI fusions by the addition of a trypsin cleavage signal immediately upstream of SFTI, and resulted in production of a 14 amino acid disulfide bonded SFTI free in the culture supernatant. To assess the potential of the secreted SFTI to protect the ability of a cytotoxic protein to kill tumor cells, we utilized a tumor-selective form of the Pseudomonas ToxA (OTG-PE38K) alone and expressed as a polycistronic construct with YebF-SFTI in the tumor-targeted Salmonella VNP20009. When we assessed the ability of toxin-containing culture supernatants to kill MDA-MB-468 breast cancer cells, the untreated OTG-PE38K was able to eliminate all detectable tumor cells, while pretreatment with trypsin resulted in the complete loss of activity. However, when OTG-PE38K was coexpressed with YebF-SFTI, activity was completely retained in the presence of trypsin. These data demonstrate that co-expression of protease inhibitors with therapeutic proteins by tumor-targeted bacteria has the potential to enhance the activity of therapeutic proteins by suppressing their degradation within a proteolytic environment.|
|Key_word||Protease inhibitors, Sunflower trypsin inhibitor (SFTI), YebF, VNP20009, tumor-targeted Salmonella, OTG-PE38K|
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