Co-Expression of a Chimeric Protease Inhibitor Secreted by a Tumor- Targeted Salmonella Protects Therapeutic Proteins from Proteolytic Degradation

• Published in: Journal of microbiology and biotechnology Vol. 28; no. 12; pp. 2079 - 2094
• Main Authors: Quintero, David, Carrafa, Jamie, Vincent, Lena, Kim, Hee Jong, Wohlschlegel, James, Bermudes, David
• Format: Journal Article
• Language: Korean
• Published: 한국미생물생명공학회 31.12.2018
• Subjects: OTG-PE38K; Protease inhibitors; sunflower trypsin inhibitor (SFTI); tumor-targeted Salmonella; VNP20009; YebF; YebF; sunflower trypsin inhibitor (SFTI); OTG-PE38K; Protease inhibitors; VNP20009; tumor-targeted Salmonella
• ISSN: 1017-7825; 1738-8872

Sunflower trypsin inhibitor (SFTI) is a 14-amino-acid bicyclic peptide that contains a single internal disulfide bond. We initially constructed chimeras of SFTI with N-terminal secretion signals from the Escherichia coli OmpA and Pseudomonas aeruginosa ToxA, 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 an individual colony of E. coli using a culturebased 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 anticancer cytotoxicity. However, when OTG-PE38K was co-expressed with YebF-SFTI, cytotoxicity was completely retained in the presence of trypsin. These data demonstrate SFTI chimeras are secreted in a functional form and 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.