Expression,Purification And Function Analysis Of Recombinant Soluble Corin

Part I. PCSK6 Mediated Activation of Soluble CorinCorin is a type II transmembrane serine protease abundantly expressed in the heart. Functional studies show that Corin is the enzyme responsible for the conversion of pro-atrial natriuretic peptide(pro-ANP) and pro-brain natriuretic peptide(pro-BNP) to mature ANP and BNP, both of which are cardiac hormones important in maintaining normal blood pressure and electrolyte homeostasis. In mice, lack of Corin prevents natriuretic peptide processing, causing salt-sensitive hypertension. In humans, Corin variants and mutations reducing Corin activity have been identified in patients with hypertension and heart failure(HF). Recently study demonstrated that Corin overexpression improves cardiac function, heart failure, and survival in mice with dilated cardiomyopathy. The results suggest that recombinant soluble Corin can be used to improve cardiac function in HF patients.Corin as a transmembrane protein, which is difficult to produce large amount of recombinant protein. Previous data indicated that the transmembrane domain is not necessary for the biological activity of Corin, thus we will choose soluble Corin for further study.Corin is synthesized as zymogen, and recently study showed that proprotein convertase subtilisin/ kextin type 6(PCSK6, also named PACE4) is the long-sought Corin activator. However, whether soluble Corin can be activated by PCSK6 remains elusive. In this study, we constructed soluble Corin and PCSK6 expression vectors, expressed recombinant protins in HEK293 cells, and checked if PCSK6 can activate soluble Corin, finally we examined the pro-ANP processing activity of activated soluble Corin.Methods:1. To construct a plasmid expressing soluble Corin, a c DNA fragment containing nucleotides 463-3219 of human Corin c DNA was amplified by PCR and inserted into the expression vector p SEC to yield the plasmid p SECsol Corin.2. Soluble Corin, PCSK6 and pro-ANP expression vectors were transfected into HEK293 cells, collected cell lysate and conditioned medium. Protein expression was determined by Western blotting.3. Soluble Corin medium was incubated with increasing amounts of PCSK6, and activated Corin and PCSK6 were analyzed by Western blotting.4. Incubated activated soluble Corin with pro-ANP to exam Corin activities.Results:1. We successfully constructed soluble Corin expression vector, and soluble Corin can be expressed in HEK293 cells.2. When soluble Corin was treated with condition medium contain increasing amount PCSK6, activated Corin protease fragments(Corin-p) can be detected and increased in a dose-dependently manner.3. Activated soluble Corin can process pro-ANP to ANP in a dose-dependently manner.Conclusions:In this study, we identified that soluble Corin can be activated by PCSK6, and exhibiting the capacity of processing pro-ANP. Our data suggested soluble Corin may become a new therapeutic strategy of heart failure. Part II. Expression, Purification and Activation of EK soluble CorinHeart failure(HF) is a life-threatening disease, afflicting tens of millions of people in the world. The morbidity and mortality in heart failure is still high. There is an unmet need for new therapy options. Corin is a trypsin-like serine protease first cloned from the heart and can convert pro-ANP to biologically active ANP, a cardiac hormone essential in controlling blood pressure and maintaining electrolyte and body fluid homeostasis. Corin deficiency may contribute to the pathogenesis of hypertension and HF. Previous studies indicated that the soluble Corin may become a new therapeutic strategy of HF.In part I, we have showed that soluble Corin can be activated by PCSK6. However, HEK 293 cells have some endogenous PCSK6, which may activate part of soluble Corin and reduce yield of recombinant protein. In addition, there is no commercial PCSK6 available. Together, it may be difficult to get high amount of activated souble Corin for futher animal study.In this part we design, express, and purify a mutant soluble Corin, EKsol Corin, that contains an enterokinase recognition sequence at the activation cleavage site, which can be activated by enterokinase.Methods:1. Plasmid contained EKCorin was used as a PCR template to generate the expression vector. The resulting plasmid, p SECEKsol Corin, encodes a soluble Corin, EKsol Corin, with the amino acid sequence DDDDK replacing the original sequence RMNKR at the conserved activation cleavage site.2. Established stable cell lines expressing EKsol Corin proteins in HEK293 cells with hygromycin(50 μg/m L), and soluble Corin levels in conditioned media were measured by an ELISA method.3. Collected conditioned media from HEK293 cells stably expressing soluble Corin proteins, and employed immobilised metal-ion affinity chromatography(IMAC) to purify soluble Corin. The purified protein was further characterized by Western blotting and Coomassie blue staining.4. To activate the recombinant soluble Corin, EKsol Corin, purified protein was incubated with increasing concentrations of recombinant EK. Samples were taken and analyzed by SDS-PAGE under reducing and non-reducing conditions followed by Western blotting using an anti-V5-antibody.5. Assessed activities of soluble Corin that had been activated by recombinant EK in processing pro-ANP by Western analysis.Results:1. We successfully constructed soluble EKCorin expression vector, which consisted of only the extracellular domain and contained EK recognition sequence.2. Screened out 25 cell lines which overexpressed EKsol Corin(>0.1 μg/m L).3. The purified protein had a purity of 55%, and purified protein can be activated totally by EK(10 units/m L) in activation buffer at 25 °C for 2 h.4. EKsol Corin that had been activated by recombinant EK was capable of processing pro-ANP in cell-based assays.Conclusions:We designed, expressed, and purified a soluble Corin named EKsol Corin that contains an EK recognition sequence at the conserved activation site. We showed that purified EKsol Corin was activated by EK and that activated EKsol Corin was capable of processing pro-ANP in cell-based assays. Together, our data provide new insight into the treatment strategies for heart failure.