Tobacco etch virus protease (TEVp) is a useful tool for removing fusion tags, but wild-type TEVp is less stable under oxidized redox state. In this work, we introduced and combined C19S, C110S and C130S into TEVp variants containing T17S, L56V, N68D, I77V and S135G to improve protein solubility, and S219V to inhibit self-proteolysis. The solubility and cleavage activity of the constructed variants in Escherichia coli strains including BL21(DE3), BL21(DE3)pLys, Rossetta(DE3) and Origami(DE3) under the same induction conditions were analyzed and compared. The desirable soluble amounts, activity, and oxidative stability were identified to be reluctantly favored in the TEVp. Unlike C19S, C110S and C130S hardly impacted on decreasing protein solubility in the BL21(DE3), but they contributed to improved tolerance to the oxidative redox state in vivo and in vitro. After two fusion proteins were cleaved by purified TEVp protein containing double mutations under the oxidized redox state, the refolded disulfide-rich bovine enterokinase catalytic domain or maize peroxidase with enhanced yields were released from the regenerated amorphous cellulose via affinity absorption of the cellulose-binding module as the affinity tag.
Background
Several fusion tags for separation handle have been developed, but the fused tag for simply and cheaply separating the target protein is still lacking.
Results
Separation conditions for the human annexin A1 (hanA1) tagged emerald green fluorescent protein (EmGFP) in Escherichia coli were optimized via precipitation with calcium chloride (CaCl2) and resolubilization with ethylenediamine tetraacetic acid disodium salt (EDTA-Na2). The HanA1-EmGFP absorbing with other three affinity matrix was detected, only it was strongly bound to heparin Sepharose. The separation efficiency of the HanA1-EmGFP was comparable with purification efficiency of the His6-tagged HanA1-EmGFP via metal ion affinity chromatography. Three fluorescent proteins for the EmGFP, mCherry red fluorescent protein and flavin-binding cyan-green fluorescent protein LOV from Chlamydomonas reinhardtii were used for naked-eye detection of the separation and purification processes, and two colored proteins including a red protein for a Vitreoscilla hemoglobin (Vhb), and a brown protein for maize sirohydrochlorin ferrochelatase (mSF) were used for visualizing the separation process. The added EDTA-Na2 disrupted the Fe–S cluster in the mSF, but it showed little impact on heme in Vhb.
Conclusions
The selected five colored proteins were efficient for detecting the applicability of the highly selective hanA1 for fusion separation and purification handle. The fused hanA1 tag will be potentially used for simple and cheap affinity separation of the target proteins in industry and diagnosis.
ObjectiveTo confirm that gut lymph purification (GLP) based on oXiris regulates monocyte activity by targeting the removal of ischemia-reperfusion injury (IRI)-induced intestinal toxic substances (ITSs) in rats.
MethodsSepsis was induced by intestinal IRI in 24 adult male Sprague-Dawley rats that were randomly divided into the control, intestinal IRI, and IRI+GLP groups. The gut lymph fluid (GLF) was drained for 180 minutes.The ITSs levels and the proliferation, apoptosis and positive expression rates of MHC-II molecules of monocytes coincubated with the GLF were detected.
ResultsEndotoxin, TNF-α, IL-4, IL-6 and IL-10 levels in the lymph and plasma of the IRI group were significantly higher than those of the control group (p < 0.01). Compared with the IRI group, GLP treatment significantly decreased the ITS levels (p < 0.05). Monocyte proliferation and the positive expression rate of MHCmolecules were significantly reduced after co-culturing with GLF upon IRI (p < 0.01), and the apoptotic rate was significantly increased (p < 0.01). However, culturing monocytes with GLP significantly enhanced the monocyte proliferation, increased the positive expression rate of MHC-monocytes (p < 0.01), and reduced the apoptotic rate (p < 0.01).
ConclusionsGLP therapy based on oXiris effectively removed ITSs from the GLF after IRI, thereby blocking the main process of multiple organ dysfunction syndrome by regulating monocyte activity.
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