Abstract
Myocardial ischemic preconditioning up-regulated protein 1 (Mipu1), a novel zinc finger protein, was originally cloned using bioinformatic analysis and 5' RACE technology of rat heart after a transient myocardial ischemia/reperfusion procedure in our laboratory. In order to investigate the functions of Mipu1, the recombinant prokaryotic expression vector pQE31-Mipu1 was constructed and transformed into Escherichia coli M15(pREP4), and Mipu1-6His fusion protein was expressed and purified. The identity of the purified protein was confirmed by mass spectrometry. The molecular mass of the Mipu1 protein was 70.03779 kDa. The fusion protein was intracutaneously injected to immunize New Zealand rabbits to produce a polyclonal antibody. The antibody titer was approximately 1:16,000. The antibody was tested by Western blotting for specificity and sensitivity. Using the antibody, it was found that Mipu1 was highly expressed in the heart and brain of rats and was localized in the nucleus of H9c2 myogenic cells. The present study lays the foundation for further study of the biological functions of Mipu1.
Highlights
During myocardial ischemia or reperfusion, many genes such as c-fos, c-jun, junB, Egr-1, and HSP70 can be upregulated [1,2,3], and some of these myocardial genes have been considered to be involved in the endogenous cardioprotection against myocardial ischemia-reperfusion injury
Most of the contaminant E. coli proteins were eluted with 100 mM imidazole, whereas the target proteins were eluted with a gradient of 200–1000 mM imidazole
The myocardial ischemic preconditioning up-regulated protein 1 (Mipu1)-6His fusion protein was induced with IPTG in soluble form (Figure 1A, lane 1), and the expression level of Mipu1-6His fusion protein without the induction of IPTG was much less (Figure 1A, lane 2)
Summary
During myocardial ischemia or reperfusion, many genes such as c-fos, c-jun, junB, Egr-1, and HSP70 can be upregulated [1,2,3], and some of these myocardial genes have been considered to be involved in the endogenous cardioprotection against myocardial ischemia-reperfusion injury. Yuan and colleagues [4] found that myocardial ischemic preconditioning up-regulated protein 1 (Mipu1) was up-regulated in rat heart after a transient myocardial ischemia-reperfusion procedure, and they cloned the full length cDNA of the Mipu gene (GenBank accession No AY221750). Jiang et al [5] reported that Mipu was a nuclear and DNA binding protein that bound a specific DNA sequence, TGTCTTATCGAA, with TCTTA as the core sequence. This suggested that Mipu might function as a transcription factor. The cDNA gene encoding rat Mipu was expressed in Escherichia coli and the Mipu16His fusion protein was purified and injected into New Zealand rabbits for the production of an anti-Mipu polyclonal antibody. The antibody was used to detect the abundance of Mipu in various rat tissues and the expreswww.bjournal.com.br sion and subcellular localization of Mipu in H9c2 myogenic cells
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