Li Yanying, Sun Yan
Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China.
J Chromatogr A. 2014 Dec 19;1373:97-105. doi: 10.1016/j.chroma.2014.11.017. Epub 2014 Nov 13.
Poly(4-vinylpyridine) (P4VP) was proposed for use as a polymeric ligand of mixed-mode chromatography (MMC) of proteins. P4VP has linear hydrophobic chains with ionizable pyridyl groups in its backbone. The polymer was coupled onto Sepharose FF gel at a pyridyl group density of 190 μmol/mL (FF-P4VP-190) by the substitution reaction of pyridyl amines with brominated Sepharose gel. Thereby the immobilized ligand possesses the intrinsic hydrophobic nature as well as the newly obtained electrostatic interaction properties endowed from the substituted positively charged pyridyl amines. The pore size distribution was measured by inverse size exclusion chromatography, and the results revealed that P4VP formed a three-dimensional layer on the matrix surface with a maximum layer depth of 4.2 nm. The adsorption isotherms of γ-globulin and bovine serum albumin (BSA) to FF-P4VP-190 were determined under varying pH values and salt concentrations to provide insights into the adsorption properties of the medium. It was found that the adsorption capacity of γ-globulin and BSA both presented an increase with pH increasing from 8.0 to 9.0. Moreover, FF-P4VP-190 exhibited stronger adsorption for BSA than γ-globulin. The higher affinity for BSA might be attributed to its more net negative charges. Protein adsorption capacities to FF-P4VP-190 decreased with increasing NaCl concentration, but still manifested moderate levels at high salt concentration such as 75 mg/mL for γ-globulin and 14 mg/mL for BSA at 0.5 mol/L NaCl and pH 9.0. The capacity decreases with increasing ionic strength, indicating the dominant role of electrostatic interactions, while the moderate capacity values at 0.5 mol/L NaCl confirmed the presence of salt-tolerant feature of FF-P4VP-190, making it function as an MMC material. Column chromatography was conducted to investigate protein elution behavior. Efficient protein recovery was achieved at mild elution conditions such as pH 4.0 for γ-globulin and pH 4.5 for BSA. The results indicate that the P4VP-based adsorbent would provide new possibilities for protein purification by MMC.
聚（4-乙烯基吡啶）（P4VP）被提议用作蛋白质混合模式色谱（MMC）的聚合物配体。P4VP具有线性疏水链，其主链上带有可离子化的吡啶基。通过吡啶胺与溴化琼脂糖凝胶的取代反应，将该聚合物以190 μmol/mL的吡啶基密度偶联到琼脂糖FF凝胶上（FF-P4VP-190）。从而，固定化配体具有内在的疏水性质以及由取代的带正电荷的吡啶胺赋予的新获得的静电相互作用特性。通过反相尺寸排阻色谱法测量孔径分布，结果表明P4VP在基质表面形成了三维层，最大层深度为4.2 nm。在不同的pH值和盐浓度下测定了γ-球蛋白和牛血清白蛋白（BSA）对FF-P4VP-190的吸附等温线，以深入了解该介质的吸附特性。发现随着pH值从8.0增加到9.0，γ-球蛋白和BSA的吸附容量均呈现增加。此外，FF-P4VP-190对BSA的吸附比对γ-球蛋白更强。对BSA的更高亲和力可能归因于其更多的净负电荷。随着NaCl浓度的增加，蛋白质对FF-P4VP-190的吸附容量降低，但在高盐浓度下仍表现出中等水平，例如在0.5 mol/L NaCl和pH 9.0时，γ-球蛋白为75 mg/mL，BSA为14 mg/mL。容量随离子强度的增加而降低，表明静电相互作用起主导作用，而在0.5 mol/L NaCl时的中等容量值证实了FF-P4VP-190具有耐盐特性，使其可作为MMC材料发挥作用。进行柱色谱法研究蛋白质洗脱行为。在温和的洗脱条件下，如γ-球蛋白在pH 4.0、BSA在pH 4.5时，实现了高效的蛋白质回收。结果表明，基于P4VP的吸附剂将为通过MMC进行蛋白质纯化提供新的可能性。
