Title |
Optimization of construct design and fermentation strategy for the production of bioactive ATF-SAP, a saporin based anti-tumoral uPAR-targeted chimera
|
---|---|
Published in |
Microbial Cell Factories, November 2016
|
DOI | 10.1186/s12934-016-0589-1 |
Pubmed ID | |
Authors |
Alfredo Errico Provenzano, Riccardo Posteri, Francesco Giansanti, Francesco Angelucci, Sopsamorn U. Flavell, David J. Flavell, Maria Serena Fabbrini, Danilo Porro, Rodolfo Ippoliti, Aldo Ceriotti, Paola Branduardi, Riccardo Vago |
Abstract |
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. 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. 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. |
X Demographics
Geographical breakdown
Country | Count | As % |
---|---|---|
Italy | 2 | 100% |
Demographic breakdown
Type | Count | As % |
---|---|---|
Members of the public | 2 | 100% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 11 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Master | 2 | 18% |
Student > Ph. D. Student | 2 | 18% |
Professor > Associate Professor | 2 | 18% |
Researcher | 2 | 18% |
Other | 1 | 9% |
Other | 0 | 0% |
Unknown | 2 | 18% |
Readers by discipline | Count | As % |
---|---|---|
Biochemistry, Genetics and Molecular Biology | 4 | 36% |
Agricultural and Biological Sciences | 3 | 27% |
Immunology and Microbiology | 1 | 9% |
Unknown | 3 | 27% |