Intein Mediated Soluble Expression And Purification System And Its Application In Recombinant Chemokine CXCL9

CXCL9 is a CXC-chemokine specifically induced by IFN-? in multiple cells.It is able to activate T-lymphocytes in immunological responses,and has been recently reported to play hematopoietic stem cell protective roles in cancer chemotherapy by us.However,CXCL9 is still difficult to prepare since it's easy to aggregate and difficult to scale up when expressed in E.coli(Escherichia coli).In order to encourage the pharmaceutical research of CXCL9,here we developed two strategies to improve its expressing solubility: a novel soluble expression process with the self-cleavable tag Zbasic-?I-CM,and the low-temperature induction.To explore the most effective fusion strategy to achieve soluble expression,we screened three candidate tags: FATT,Fh8,and Zbasic,being fused at the N-terminus of ?I-CM-CXCL9.Zbasic-?I-CM-CXCL9 showed higher yield of product according to densitometry quantification.Adjustment of induction temperature also can effectively affect the solubility or yield of target protein expression in E.coli.We expressed CXCL9 alone at 25°C in comparison with Zbasic-?I-CM tag at 37°C to evaluate their efficiency,product quality and yield.CXCL9 was successfully purified from bacteria supernatant with SP Sepharose cation-exchange chromatography and a second polishing step of CHT I(CHT? Ceramic Hydroxyapatite Type I)column.Compared with traditional inclusion bodies expression,we demonstrated that Zbasic-?I-CM mediated soluble expression achieved excellent recovery,manipulatory feasibility and scalability.All products were identified to be with correct molecular weight of 12190 Da and with >97% purity by SEC-HPLC.Based on a HUVEC(human umbilical vein endothelial cell)inhibitory proliferation assay,the products showed significant biological activity.With improved purification efficiency and resources consumption,Zbasic-intein mediated soluble expression was convinced to be an advanced novel process for CXCL9 production.Our work also illustrated the application potential of the approach in the industrial manufacturing of biopharmaceuticals from E.coli expression system.