Recombination And Purification Of DDAH2 Protein And Its Function Study

BACKGROUND Cardiovascular diseases,as the most common diseases of human being,have become one of the leading causes of death in developed countries as well as in many developing countries.Endothelial dysfunction, mainly characterized as reduction of nitric oxide(NO)-mediated endothelium-dependent vasodilatation,tends to precede diversed clinical manifestations of most cardiovascular diseases and to be an early pathological event.A substantial body of evidence suggests that elevated levels of endogenous nitric oxide synthase(NOS)inhibitor asymmetric dimethylarginine(ADMA)is involved in the mechanisms of endothelial dysfunction for cardiovascular diseases.Contribution of elevated endogenous ADMA to endothelial dysfunction predominantly lies in the decreased expression or activity of dimethylarginine dimethylaminohydrolase 2 (DDAH2),which is ADMA's main metabolic enzyme and extensively located in endothelial cells.DDAH is composed of 2 isoforms,DDAH1 and DDAH2. DDAH1 is typically found in tissues of expressing neuronal NOS,whereas DDAH2 predominates in cardiovascular tissues of expressing endothelial NOS. So DDAH2 play a pivotal role in modulating ADMA metabolism in cardiovascular system.However,no medicine has ever been found to directly increase expression and activity of DDAH2.Until recently,studies have identified a protein domain,namely protein transduction domain(PTD),from human immunodeficiency virus tans-activator transcription protein(HIV-1 Tat),which can efficiently conduct peptides or proteins into living cell,thus playing a role of protein transduction.The objectives of this study are firstly to fuse Tat PTD to the N-terminal of DDAH2 protein,so as to enable DDAH2 protein to possess the ability of go through cell membrane and produce effects inside cells.To go a step further,we will also study effects of cardiovascular risks such as high glucose,homocystein and palmitic acid on expressions of DDAH1 and DDAH2 protein and activities of DDAH in cultured human umbilical vein endothelial cells(HUVEC-12)pretreatmented with or without Tat-hDDAH2 protein.Our work will provide a potential therapeutic method of treating cardiovascular diseases with recombined protein products.Moreover, we will prepare the anti-serum from New Zealand rabbits against recombination of GST-hDDAH2 fusion protein for the detection of DDAH2 protein expression by western blotting in this study.METHODS①Construction of recombination prokaryotic expression vector of pGEX-6p-1-Tat-hDDAH2:Taken the plasmid pcDNA3.1-hDDAH2 as template,we amplified Tat-hDDAH2 gene using the forward and reverse primers of Tat-hDDAH2,and ligated it with T-vector to construct pGEM-Tat-hDDAH2 plasmid.The plasmid and prokaryotic expression vector pGEX-6p-1 were digested with SalⅠand BamHⅠfor over night,respectively. We ligated the incised plasmid and vector to recombinate the vector of pGEX-6p-1-Tat-hDDAH2,which were then transformed into host bacteria E coli BL21 to induce maximal expression of the targeted gene and produce soluble fusion proteins in optimized conditions.②Purification of Tat-hDDAH recombination protein:First,recombinant proteins in form of inclusion bodies were dissolved in urea solution(8 M)and then after renaturation and dialysis, the renaturated protein GST-Tat-hDDAH was achieved.Nextly,the soluble GST-Tat-hDDAH protein in the supernatant of the bacterial lysate and the renaturated protein collected from the inclusion bodies were thoroughly mixed with Glutathione Sepharose 4B and then get purified by the affinity adsorption. Finally,the purified GST-Tat-hDDAH2 protein was incised by PreScission protease,and recombinant protein Tat-hDDAH2 was obtained.③Preparation of anti-hDDAH2 anti-serum:After recombination and transformation of pGEX-6p-1-hDDAH2 vector,the host bacteria were induced to express and produce a great quantity of inclusion bodies containing GST-hDDAH2 protein.The inclusion bodies washed by 2%sodium deoxycholate solution was thoroughly emulsified with the adjuvant and used to immunize New Zealand rabbits by subcutaneous injections.After the anti-DDAH2 polyclonal antibody was prepared,its titer and specificity were determined by ELISA and western blotting,respectively.We used it to detect expression of DDAH2 protein in the present study.④Functional study of Tat-hDDAH2 recombination protein:The human umbilical venous endothelial cell line HUVEC-12 were cultured and incubated with different risk factors of cardiovascular diseases,including high glucose,homocysteic acid and free fatty acid such as palmitic acid(PA)in the absence or presence of Tat-hDDAH2 protein for 16～48 h.Expressions of DDAH1 and DDAH2 proteins,as well as activities of DDAH,were detected to review the important role of DDAH2 in endothelial function and to explore the potential protective effects of Tat-hDDAH2 protein against these risk factors.RESULTS①As identified by PCR,digestion and sequencing,prokaryotic expression vectors of both pGEX-6p-1-Tat-hDDAH2(Fig.1～3)and pGEX-6p-1-hDDAH2(Fig.4)were successfully constructed and then transformed to E.coli BL21,in which were efficiently amplified to express soluble fusion proteins of GST-Tat-hDDAH2(Fig.5～7)and GST-hDDAH2 (Fig.9～11)by using a low concentration of IPTG(0.05 mM),at low temperature(25℃)and for a extended cultural time(8 h).However,in prokaryotic cells,the amplified fusion proteins occur in the form of insoluble inclusion bodies predominantly.So the inclusion bodies were denaturated, renaturated,purified and incised by PreScission protease to obtain the Tat-hDDAH2(Fig.8)and DDAH2(Fig.12)recombination proteins finally.②One liter of bacterial culture solution containing pGEX-6p-1-Tat-hDDAH2 and pGEX-6p-1-DDAH2 plasmids was centrifuged at 12000 rpm,4℃,for 10 min.The supernatant was discarded and the pellet was resuspended in 100 ml PBS.After spallation by ultrasound,the resuspension solution was centrifuged at 12000 rpm,4℃for 10 min to separate the soluble protein in supernatant and insoluble inclusion bodies.After the inclusion bodies were denaturated, renaturated,the renaturated protein collected from the inclusion bodies and the soluble fusion protein in the supernatant were purified by affinity adsorption with Glutathione Sepharose 4B to get 1.545 mg of purified GST-Tat-hDDAH2 protein and 1.735 mg of GST-hDDAH2 fusion protein(Tab.1～2).These proteins were incised by PreScission protease to obtain 1.085 mg of recombinant Tat-hDDAH2 protein and 1.154 mg of recombinant hDDAH2 protein(Tab.1～2).③After immunizating New Zealand rabbits with inclusion bodies containing GST-hDDAH2 for 10 weeks,we obtained the polyclonal antibody against human DDAH2 protein in a titer of 1:10000 by centrifuging the blood from the rabbits.In addtion,we used this anti-serum for measurement of recombinant DDAH2 protein,recombinant Tat-hDDAH2 proteins and expression of DDAH2 protein in HUVEC-12.④Incubation of HUVEC-12 with 0.1～1μM Tat-hDDAH2 for 60 min,the expression of DDAH2 and activity of DDAH in the HUVEC-12 were examined to see the dose-response of Tat-hDDAH2.The results showed that Tat-hDDAH2 upregulated DDAH2 expression and enhanced DDAH activity in HUVEC-12 in a dose-dependent manner(Fig.15).Moreover,the time course for Tat-hDDAH2 action was checked by treatment of endothelial cells with 1μM Tat-hDDAH2 for 5～60 min.Similiarly,Tat-hDDAH2 upregulated DDAH2 expression and enhanced DDAH activity in HUVEC-12 in a time-dependent manner.However,incubation of endothelial cells with 10～30 mM glucose for 48 h or with 1～3 mM Hcy and 0.1～0.5 mM PA for 16 h,not only DDAH2 expression was suppressed(Fig.17,19)but also DDAH activity was inhibited (Fig.18,20)in a dose-dependent manner.Pretreatment with 0.1～0.5μM Tat-hDDAH2 for 1 h could protect endothelial cells from the deleterious effect of these risk factors for cardiovascular deseases on DDAH2 expression and DDAH activity.CONCLUSIONS①The construction of prokaryotic expression vectors pGEX-6p-1-Tat-hDDAH2 and pGEX-6p-1-Tat-hDDAH2 was achieved.②The prokaryotic expression vector of pGEX-6p-1-Tat-hDDAH2 and pGEX-6p-1-Tat-hDDAH2 was transformed into Escherichia coli BL21.After purification,GST-hDDAH2 and GST-Tat-hDDAH2 recombination proteins in high purity were obtained.Further experiments confirmed that Tat-hDDAH2 protein can not only cross biological membranes efficiently but also prossess favourable function of up-regulating the DDAH2 expression and DDAH activity in endothelial cell in time- and dose-dependent manner.③Polyclonal antibody agaist hDDAH2 with a high potency was prepared successfully by immunizating New Zealand rabbits with inclusion bodies containing GST-hDDAH2.This antibody can be used to detect recombination GST-hDDAH2,Tat-hDDAH2 hDDAH2 protein and DDAH2 expressing in human umbilical vein endothelial cells.④This study showed that reductions of DDAH activity induced by cardiovascular risk factors such as high glucose, homocystein and free fatty acids attributed to down-regulation of expression of DDAH2.Tat-hDDAH2 can protect endothelial cells from the deleterious effect of these risk factors for cardiovascular deseases on DDAH2 expression and DDAH activity in in time- and dose-dependent manner.