Journal
MICROBIAL CELL FACTORIES
Volume 15, Issue -, Pages -Publisher
BMC
DOI: 10.1186/s12934-016-0589-1
Keywords
Targeted therapy; Saporin; Yeast expression system; Fed-batch production; Chimeric fusions; Ribosome inactivating proteins
Categories
Funding
- Leukaemia Busters
- University of L'Aquila
- Italian Ministry of Health [GR-2011-02351220]
- FAR (Fondo di Ateneo per la Ricerca) of the University of Milano-Bicocca
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Background: The big challenge in any anti-tumor therapeutic approach is represented by the development of drugs selectively acting on the target with limited side effects, that exploit the unique characteristics of malignant cells. The urokinase (urokinase-type plasminogen activator, uPA) and its receptor uPAR have been identified as preferential target candidates since they play a key role in the evolution of neoplasms and are associated with neoplasm aggressiveness and poor clinical outcome in several different tumor types. Results: To selectively target uPAR over-expressing cancer cells, we prepared a set of chimeric proteins (ATF-SAP) formed by the human amino terminal fragments (ATF) of uPA and the plant ribosome inactivating protein saporin (SAP). Codon-usage optimization was used to increase the expression levels of the chimera in the methylotrophic yeast Pichia pastoris. We then moved the bioprocess to bioreactors and demonstrated that the fed-batch production of the recombinant protein can be successfully achieved, obtaining homogeneous discrete batches of the desired constructs. We also determined the cytotoxic activity of the obtained batch of ATF-SAP which was specifically cytotoxic for U937 leukemia cells, while another construct containing a catalytically inactive mutant form of SAP showed no activity. Conclusion: Our results demonstrate that the uPAR-targeted, saporin-based recombinant fusion ATF-SAP can be produced in a fed-batch fermentation with full retention of the molecules selective cytotoxicity and hence therapeutic potential.
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