A simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease.

Recombinant protein purification remains to be a major challenge in biotechnology and medicine. In this paper we report a simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease (TEVp). After construction of an N-terminal ELK16 peptide fusion expression vector, we expressed ELK16-TEVp fusion protein in E. coli. SDS-PAGE analysis showed that ELK16-TEVp was expressed as active protein aggregates which could be purified to 91% purity with 92% recovery by centrifugation in the presence 0.5% Triton X-100. By using His-tagged bovine interferon-γ (His-BoIFN-γ) as the substrate, we demonstrated that EKL16-TEVp had a protease activity of 1.3 × 10(4) units/mg protein with almost 100% cleavage efficiency under the optimized conditions. More importantly, EKL16-TEVp could be removed from the cleavage reaction by single-step centrifugation. After removing the His-tag by nickel-conjugated agarose bead absorption, the recombinant BoIFN-γ (rBoIFN-γ) was purified to 98.3% purity with 63% recovery. The rBoIFN-γ had an antiviral activity of 1.6 × 10(3) units/mg protein against vesicular stomatitis virus. These data suggest that ELK16-TEVp may become a universal tool for recombinant protein purification.

Single-step purification of recombinant proteins using elastin-like peptide-mediated inverse transition cycling and self-processing module from Neisseria meningitides FrpC.

Purification of recombinant proteins is a major task and challenge in biotechnology and medicine. In this paper we report a novel single-step recombinant protein purification system which was based on elastin-like peptide (ELP)-mediated reversible phase transition and FrpC self-processing module (SPM)-mediated cleavage. After construction of a SPM-ELP fusion expression vector, we cloned the coding sequence for green fluorescent protein (GFP), the Fc portion of porcine IgG (pFc) or human ß defensin 3 (HBD3) into the vector, transformed the construct into Escherichia coli, and induced the fusion protein expression with IPTG. The target-SPM-ELP fusion proteins GFP-SPM-ELP, Fc-SPM-ELP and HBD3-SPM-ELP were expressed in a soluble form and efficiently purified from the clarified cell extracts by two rounds of inverse transition cycling (ITC). Under the optimized conditions, the SPM-mediated cleavage efficiencies for the three fusion proteins ranged from 92% to 93%. After an additional round of ITC, the target proteins GFP, pFc and HBD3 were recovered with purities ranging from 90% to 100% and yields ranging from 1.1 to 36mg/L in shake flasks. The endotoxin levels in all of the three target proteins were <0.03EU/mg. The three target proteins were functionally active with the expected molecular weights. These experimental results confirmed the high specificity and efficiency of SPM-mediated cleavage, and suggested the applicability of SPM-ELP fusion system for purification of recombinant proteins.

Effects of Tongguan Capsule on post-myocardial infarction ventricular remodeling and cardiac function in rats.

OBJECTIVE: To observe the effects of Tongguan Capsule (TGC) on post-myocardial infarction ventricular remodeling and heart function in rats. METHODS: A rat model of acute myocardial infarction (AMI) was established by coronary ligation. Experimental rats were randomized to 4 groups including three model groups (Group A: captopril 5 mg/kg * day, n=7; Group B: TGC 10 g/kg * day, n=7; and Group C: placebo, n=8), and a sham-control group (Group D: blank control, n=6). Animals were treated for 4 weeks. The cardiac function of rats was assessed at the end of the experiment based on left ventricular ejection fraction (LVEF) and left ventricular short axis fractional shortening (LVFS) detected by colored echocardiography; meanwhile, the condition of ventricular remodeling was observed through the levels of left ventricular mass (LVM), plasma aldosterone (ALD), myocardial angiotensin II (Ang II) and myocardial collagen measurements. RESULTS: At the end of the experiment, LVEF and LVFS in Group A and B were improved significantly, while those in Group C were unchanged, the LVEF in Group A, B, C, and D was 0.57+/-0.46, 0.61+/-0.08, 0.36+/-0.55 and 0.76+/-0.02, respectively; and their LVFS was 0.31+/-0.52, 0.34+/-0.04, 0.23+/-0.57 and 0.45+/-0.03, respectively. The difference was statistically significant when comparing the two indexes in Group A and B with those in Group C and D (P<0.05). LVM, levels of plasma ALD and myocardial Ang II were lower in Group A and B than in Group C, but a comparison between Group A and B showed an insignificant difference in lowering LVM and ALD, while the lowering of Ang II was more significant in Group B than in Group A (754.7 +/- 18.7 pg/mL vs 952.6+/-17.6 pg/mL, P<0.05). Morphological examination showed that in Group A and B the swollen myocardial cells had shrunk, with regularly arranged myocardial fibers and decreased collagen proliferation, but the improvements in Group B were more significant. CONCLUSION: TGC could markedly improve the post-infarction ventricular remodeling and cardiac function in rats, showing that the efficacy was better than or equal to that of captopril.