Article
Protein therapy for treatment of cerebral vasospasm after subarachnoid hemorrhage using hemeoxygenase-1-fused 11R protein in rats
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Authors
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T. Ogawa
- Neurochirurgische Klinik, Klinikum der Heinrich-Heine-Universität, Düsseldorf
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D. Hänggi
- Neurochirurgische Klinik, Klinikum der Heinrich-Heine-Universität, Düsseldorf
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H.-J. Steiger
- Neurochirurgische Klinik, Klinikum der Heinrich-Heine-Universität, Düsseldorf
| Published: | May 20, 2009 |
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Outline
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Objective: By conjugating 10–20 amino acid peptides, which is called a “protein transduction domain (PTD)”, several proteins can be transduced directly, harmlessly and effectively into all kinds of cells. This method is called “protein therapy”. Eleven sequential arginine arrangements, that is, “11R” is one of the most excellent PTD. Previously, we examined and proved that 11R-fused enhanced green fluorescent protein (11R-EGFP) immediately and effectively penetrated into all layers of the rat basilar artery (BA), especially into the tunica media (smooth muscle layer). The current project is to examine the actual therapeutic effect of 11R-fused heme oxygenase-1 (HO-1) protein, which is one of the strongest vasodilating proteins. At first, we tried to construct 11R fused HO-1 protein.
Methods: Full-length human HO-1 cDNA, which encodes a therapeutic protein, was subcloned into two-digested pET-21a(+)-11R vector. The constructed plasmids were transformed into BL21-DE3 Escherichia coli cells. The proteins are expressed in the cells by induction with isopropyl-1 thio-β-D-galactopyranoside (IPTG). After collection the pellets were exposed to ultrasound and denatured in lysis buffer containing 20 mM 2-[4-(2-Hydroxyethyl)-1-piperadinyl] ethane-sulfonic acid (HEPES) (pH 8.0), 100 mM NaCl, 8 M urea, and 20 mM imidazole. The denatured soluble fraction was applied to a column of Ni- nitriloacetic acid (NTA) agarose to purify His-tagged 11R-HO-1 proteins.
Results: After incubation, each sample was loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and the gel was then stained with Coomassie blue (n=4). The staining revealed a robust 11R-HO-1 protein expression (32kDa). In addition to that, a strong 11R-HO-1 protein expression could be achieved by Western blotting analysis (n=6).
Conclusions: We succeeded in expressing 11R fused HO-1 protein. As a next step, we will examine transduction efficacy of 11R-HO-1 protein in rat BAs and whether protein transduction of 11R-HO-1 ameliorates cerebral vasoconstriction after subarachnoid hemorrhage (SAH) in rat SAH model.
