br Materials and methods br

Materials and methods
Results and discussion During recent years, recombinant ASK1 has been obtained by expensive baculovirus-insect cell Heparin sodium systems [20], [27]. In the present study, we constructed recombinant ASK1 and expressed in active form at high level in E. coli. Human ASK1 catalytic domain (residues 649–946) was amplified from human cDNA and subcloned into the expression vector pET-42a. The protein was expressed as N-terminal GST-tagged and C-terminal His6-tagged fusion protein by using Escherichia coli Rosetta strain. Sequencing of cloned DNA showed that the fragments had been correctly inserted and the sequences were identical to those published (Accession No. BC088829.1). Expression was achieved using IPTG and auto-induction procedures. The observed molecular weight of the protein obtained corresponded to that predicted (60.0 kDa). The production level of recombinant ASK1 was around 0.47 g/L E. coli culture utilizing auto-induction protocol and 0.3 g/L after IPTG induction. Using auto-induction we have obtained more total target fusion protein, but significantly less yield of soluble target fusion protein than using IPTG induction. ASK1-GST fusion protein has mainly accumulated in the inclusion bodies form. An expression of ASK1-GST fusion protein by auto-induction method requires future optimization for increasing level of soluble proteins production (optimization of nutrient composition of media or/and decreasing temperature for E. coli cultivation). Induction with IPTG is preferred method for production of ASK1-GST fusion protein in soluble form. Utilizing the rapid and effective one-step on column purification method, protein was obtained with high purity and biological activity (Fig. 1). The final protein product was about 20% active. Thus, we have expressed ASK1 in E. coli in soluble form and propose efficient method for obtaining it in a purified form. All the relevant data on the protein expression and purification experiments are summarized in Table 1. Enzyme activity of recombinant ASK1 was determined using Kinase-Glo® Plus Luminescent Kinase Assay which provides a homogeneous high-throughput screening method for measuring kinase activity by quantitating the amount of ATP remaining in solution following a kinase reaction. The luminescent signal is correlated with the amount of ATP present and inversely correlated with the amount of kinase activity. The enzyme activity curves for ASK1 catalytic subunits obtained using IPTG induction and auto-induction protocol are shown in Fig. 2. The optimal amount of protein kinase to use in subsequent compound screens is an amount that produces luminescence values in the linear range of the kinase titration curve. Therefore, for inhibitor study the optimal concentration of ASK1 is in the range of 200–700 ng. The inhibitor study was performed using well-established ASK1-specific inhibitor NQDI-1 [22]. For determination of inhibitor activity, NQDI-1 was included in the ASK1 activity tests at final concentrations ranging from 0 to 10 μM (Fig. 3). The IC50 for NQDI-1 is about 3 μM. This value is the same that was determined using p32 kinase assay [22].
Acknowledgements This work was supported by a grant from the National Academy of Sciences of Ukraine (0112U000254).
Introduction Heart failure refers to the hypofunction of the heart associated with pathological left ventricular remodeling (Gerdes, 2002). The main influencing factor is hypertension, which accounts for 39% of male heart failure events and 59% of female cases (Kannel, 2000). Hypertensive ventricular remodeling includes changes in the size, form and function of the heart and further development may cause the deterioration of clinical symptoms. That is the pathological basis of the occurrence and development of heart failure and influences the outcome and prognosis of the diseases. Therefore, research on the pathogenesis and treatment strategies for hypertensive ventricular remodeling is of great significance.