1.Presence of imidazole in loading buffer prevents formation of free radical in immobilized metal affinity chromatography and dramatically improves the recovery of herpes simplex virus type 1 gene therapy vectors.
Jiang C;Glorioso JC;Ataai M J Chromatogr A. 2006 Jul 14;1121(1):40-5. Epub 2006 Jun 2.
We have recently shown that immobilized metal affinity chromatography (IMAC) is an effective technique for purification of herpes simplex virus type 1 (HSV-1) gene vector engineered to display cobalt affinity tag on the envelope. However, the tagged HSV-1 viruses were severely inactivated by oxidative hydroxyl free radicals when crude HSV-1 supernatant was applied on an immobilized cobalt column and eluted by a low pH buffer. Furthermore, we have reported that virus inactivation could be prevented by inclusion of high concentration of ascorbate in chromatographic mobile phase. In this paper we report that when elution of bound virus was attempted by inclusion of imidazole in elution buffer, rather than lowering the pH of elution buffer, similar inactivation was also observed. The results also demonstrated that virus inactivation was dramatically reduced by inclusion of 20mM imidazole in the loading buffer. Electron spin resonance (ESR) experiments suggest that imidazole prevents hydroxyl free radical generation from the cobalt complexes. This is the first report describing the role of imidazole in preventing free radical formation in an IMAC column. From a practical stand point, our results imply that inclusion of appropriate amount of imidazole in the loading buffer is an effective strategy for improving the recovery yield of active products and for enhancing product quality during IMAC purification.
2.Chitosan/cellulose-based beads for the affinity purification of histidine-tagged proteins.
Shao M;Xiu L;Zhang H;Huang J;Gong X Prep Biochem Biotechnol. 2018 Apr 21;48(4):352-360. doi: 10.1080/10826068.2018.1446154. Epub 2018 Apr 10.
Chitosan/cellulose-based beads (CCBs) for the affinity purification of histidine-tagged proteins were prepared from chitosan/cellulose dissolved in ionic liquid as a solvent, and their structures were characterized by Fourier transform infrared spectroscopy, transmission electron microscopy, and thermogravimetric analysis. The affinity purification was used to separate hexahistidine-tagged (his-tagged) enhanced green fluorescent protein (EGFP) from Escherichia coli. The results showed that Zn;2+;-CCB exhibited more specific adsorption capacity toward the target protein compared with Ni;2+;-CCB and Cu;2+;-CCB. The maximum adsorption of EGFP was 1.84 mg/g of Zn;2+;-CCB, with 90% purity under the optimized conditions (ionic strength (1.0 M NaCl), pH (7.2) and imidazole concentration (500 mM)). In addition, a regeneration method for the sorbent was further developed by washing with ethylenediaminetetraacetic acid disodium and then reimmobilizing with metal ions. This technique is an alternative method for the purification of his-tagged proteins, making the process more economical, fast, stable, and large batch.
3.Photocatalytic reduction of CO
Zhou SS;Liu SQ Photochem Photobiol Sci. 2017 Oct 11;16(10):1563-1569. doi: 10.1039/c7pp00211d.
Cocatalysts are vital for improving photocatalytic activity. Incorporating nitrogen atoms on a graphene frame using an imidazole cycle resulted in a new N-doped graphene (denoted as ING). Cerium(iv) oxide (CeO;2;) nanoparticles were dispersed on ING sheets, producing an ING/CeO;2; hybrid material. The ING/CeO;2; hybrid material was characterized using X-ray diffraction, transmission electron microscopy, Raman spectroscopy, ultraviolet-visible diffuse reflectance spectroscopy and surface photovoltage spectroscopy. Copper(ii) ions [Cu(ii)] were adsorbed on the ING/CeO;2; hybrid material to directly form Cu(ii)/ING/CeO;2;, which could capture the photogenerated electrons to reduce carbon dioxide (CO;2;) to methanol (CH;3;OH) under incident light irradiation. The results showed that the yield from reducing CO;2; to CH;3;OH during the photocatalytic process using Cu(ii)/ING/CeO;2; as the photocatalyst approached 385.8 μmol g;-1; cat. h;-1;, whereas the yield was only 3.57 μmol g;-1; cat. h;-1; using ING/CeO;2; as the photocatalyst. This shows that the Cu(ii) ions play a vital role during photocatalytic reduction of CO;2; by forming copper(i) ions [Cu(i)]. The percentage of ING in the ING/CeO;2; hybrid material was investigated, and the results indicated that 3.