Optimized Purification Of A Active Peptide Of Shark Angiogenesis Inhibition Factor(aSAIF) And Its Mechanism Of Tumor Angiogenesis Inhibition

Object：To optimize the conditions of high density fermentation and purification ofrecombinant active peptide of shark angiogenesis inhibition factor(aSAIF), and try toexplore its mechanism of anti-angiogenesis and anti-tumor.Methods: Find out the best IPTG concentration and induction time in5L culturefermenter. The recombinant protein was purified with ion-exchange chromatographyfirstly by optimized isocratic elution. The enriched fusion protein was attached toNi-NTA column with His-tag. His-SUMO tag was digested with optimizedconditions for SUMO proteases. aSAIF was obtained by Ni-NTA chromatography. Itsbiological ability for anti-angiogenesis was detected by WST-8test. Flow cytometrywas used to detect the effect of aSAIF on cell death and cell cycle, and mRNA expressionmicroassay to analysis the possible mechanism of anti-angiogenesis and anti-tumor ofaSAIF.Results: The best fermentation condition was confirmed as1mmol/L IPTG added3hours later after inoculation. The main parameters of purification procedure wereoptimized as follows: ion exchange chromatographic column was eluted with0.2mol/L sodium chloride and affinity chromatographic column with250mmol/Limidazole at pH7.4. The aSAIF-SUMO was digested with the ratio of of SUMOprotease to fusion protein as1:480for1h. The released aSAIF was abtained byNi-affinity chromatography to be95%of purity.The recmbianant aSAIF wasdemonstrated to have inhibitory ability for angiogenesis, but not for cell apoptosis orcell cycle. There were some genes, which involving in cellular movement,development, growth and proliferation,and death, upregulated and downregulated attranscription levels. Conclusion: The process for aSAIF fermentation and purification was developedsuccessfully, which is a crucial basis for subsequent large-scale production and studieson its biological mechanism.