title: Optimization of construct design and fermentation strategy for the production of bioactive ATF-SAP, a saporin based anti-tumoral uPAR-targeted chimera.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Istituto Biologia e Biotecnologia Agraria, CNR, Milan, Italy.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Errico Provenzano
authors.affiliation: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Milan, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Posteri
authors.affiliation: Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.|Interuniversity Consortium INBB Biostructures and Biosystems National Institute, Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Giansanti
authors.affiliation: Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Angelucci
authors.affiliation: The Simon Flavell Leukaemia Research Laboratory, (Leukaemia Busters), Southampton General Hospital, Southampton, UK.
authors.forename: Sopsamorn U
authors.initials: SU
authors.lastname: Flavell
authors.affiliation: The Simon Flavell Leukaemia Research Laboratory, (Leukaemia Busters), Southampton General Hospital, Southampton, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Flavell
authors.affiliation: Istituto Biologia e Biotecnologia Agraria, CNR, Milan, Italy.
authors.forename: Maria Serena
authors.initials: MS
authors.lastname: Fabbrini
authors.affiliation: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Milan, Italy.
authors.forename: Danilo
authors.initials: D
authors.lastname: Porro
authors.affiliation: Department of Life, Health and Environmental Sciences, University of L'Aquila, L'Aquila, Italy.|Interuniversity Consortium INBB Biostructures and Biosystems National Institute, Rome, Italy.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Ippoliti
authors.affiliation: Istituto Biologia e Biotecnologia Agraria, CNR, Milan, Italy.
authors.forename: Aldo
authors.initials: A
authors.lastname: Ceriotti
authors.affiliation: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Milan, Italy. paola.branduardi@unimib.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Branduardi
authors.affiliation: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Milan, Italy. vago.riccardo@hsr.it.|Urological Research Institute, Division of Experimental Oncology, IRCCS San Raffaele Hospital, Milan, Italy. vago.riccardo@hsr.it.|Università Vita-Salute San Raffaele, Milan, Italy. vago.riccardo@hsr.it.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Vago
mesh_terms: D019149:Bioreactors; D004354:Drug Screening Assays, Antitumor; D005285:Fermentation; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D054789:Ribosome Inactivating Proteins, Type 1 / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D000076985:Saporins; D020298:U937 Cells; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
chemical_list: D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins; D014568:Urokinase-Type Plasminogen Activator
keywords: Chimeric fusions; Fed-batch production; Ribosome inactivating proteins; Saporin; Targeted therapy; Yeast expression system