PbrD is a lead (II) binding protein encoded by the pbr lead resistance operon found exclusively in Cupriavidus metallidurans CH34. Its ability to sequester Pb(II) shows potential for it to be developed as a biosorbent for Pb in the bioremediation of contaminated wastewaters. In this study the pbrD gene from C. metallidurans CH34 was transformed and overexpressed in Escherichia coli BL21 (DE3) using the pET32 Xa/Lic vector. Optimal expression of recombinant (r)PbrD (∼50 kDa) was achieved post-induction with IPTG within inclusion bodies (IBs). Inclusion bodies were solubilised by denaturation and purified by Ni-NTA affinity chromatography. The purified denatured protein containing the N-terminal Trx•Tag™, His•Tag® and S®Tag™ was refolded in vitro via dialysis to a biologically functional form. Circular dichroism spectra of refolded rPbrD-fusion protein indicated a high degree of turns, β-sheets and 310 helices content and tryptophan fluorescence showed a structural conformational change in the presence of Pb(II). Refolded rPbrD-fusion protein bound 99.7% of Pb(II) when mixed with lead nitrate in ten-fold increasing concentrations. Adsorption isotherms including Langmuir, Freundlich, Temkin and Dubinin-Radushkevich models were applied to determine the biosorption mechanism. A biologically functional rPbrD-fusion protein has potential application in the development of a biosorbent for remediation of Pb(II) from wastewater.

中文翻译：

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功能性细菌金属伴侣PbrD融合构建体作为Pb（II）潜在生物吸附剂的重组表达和纯化。

PbrD是一种铅（II）结合蛋白，由金属铜尿嘧啶铜CH34中的pbr铅抗性操纵子编码。它具有螯合Pb（II）的能力，显示出有潜力将其开发为Pb在被污染废水的生物修复中的生物吸附剂。在这项研究中，使用pET32 Xa / Lic载体在大肠杆菌BL21（DE3）中转化了金属利德氏梭菌CH34的pbrD基因并在其中过表达。用IPTG诱导后在包涵体（IBs）中实现重组（r）PbrD（〜50 kDa）的最佳表达。包涵体通过变性溶解并通过Ni-NTA亲和层析纯化。含有N末端Trx•Tag™，His•Tag®和S®Tag™的纯化变性蛋白通过透析在体外重折叠为生物学功能形式。重新折叠的rPbrD-融合蛋白的圆二色性光谱表明存在很高的转角，β-折叠和310个螺旋含量，色氨酸荧光表明在Pb（II）存在下结构构象变化。当与硝酸铅混合以十倍递增的浓度混合时，重新折叠的rPbrD-融合蛋白结合了99.7％的Pb（II）。吸附等温线包括Langmuir，Freundlich，Temkin和Dubinin-Radushkevich模型用于确定生物吸附机理。具有生物学功能的rPbrD融合蛋白在开发用于修复废水中Pb（II）的生物吸附剂方面具有潜在的应用。与硝酸铅混合以10倍的浓度增加时，有7％的Pb（II）。吸附等温线包括Langmuir，Freundlich，Temkin和Dubinin-Radushkevich模型用于确定生物吸附机理。具有生物学功能的rPbrD融合蛋白在开发用于从废水中修复Pb（II）的生物吸附剂方面具有潜在的应用前景。与硝酸铅混合以10倍的浓度增加时，有7％的Pb（II）。吸附等温线包括Langmuir，Freundlich，Temkin和Dubinin-Radushkevich模型用于确定生物吸附机理。具有生物学功能的rPbrD融合蛋白在开发用于从废水中修复Pb（II）的生物吸附剂方面具有潜在的应用前景。