期刊论文详细信息
Microbial Cell Factories
Optimization of construct design and fermentation strategy for the production of bioactive ATF-SAP, a saporin based anti-tumoral uPAR-targeted chimera
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
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【 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

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