Optimization of construct design and fermentation strategy for the production of bioactive ATF-SAP, a saporin based anti-tumoral uPAR-targeted chimera

Microbial Cell Factories

Research

Francesco Angelucci¹  Rodolfo Ippoliti²  Francesco Giansanti²  Paola Branduardi³  Danilo Porro³  Riccardo Posteri³  Riccardo Vago⁴  Aldo Ceriotti⁵  Alfredo Errico Provenzano⁵  Maria Serena Fabbrini⁵  David J. Flavell⁶  Sopsamorn U. Flavell⁶ 

[1] Department of Life, Health and Environmental Sciences, University of L’Aquila, L’Aquila, Italy;Department of Life, Health and Environmental Sciences, University of L’Aquila, L’Aquila, Italy;Interuniversity Consortium INBB Biostructures and Biosystems National Institute, Rome, Italy;Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Milan, Italy;Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Milan, Italy;Urological Research Institute, Division of Experimental Oncology, IRCCS San Raffaele Hospital, Milan, Italy;Università Vita-Salute San Raffaele, Milan, Italy;Istituto Biologia e Biotecnologia Agraria, CNR, Milan, Italy;The Simon Flavell Leukaemia Research Laboratory, (Leukaemia Busters), Southampton General Hospital, Southampton, UK;

关键词: Targeted therapy;    Saporin;    Yeast expression system;    Fed-batch production;    Chimeric fusions;    Ribosome inactivating proteins;   

DOI  :  10.1186/s12934-016-0589-1

 received in 2016-04-26, accepted in 2016-11-03,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundThe 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.ResultsTo 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.ConclusionOur 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.

【 授权许可】

CC BY   
© The Author(s) 2016