1. Recombinant expression and purification of a functional bacterial metallo-chaperone PbrD-fusion construct as a potential biosorbent for Pb(II).
- Author
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V K, I A, Hw D, and K K
- Subjects
- Cupriavidus genetics, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Cupriavidus chemistry, Lead chemistry, Metalloproteins biosynthesis, Metalloproteins chemistry, Metalloproteins genetics, Metalloproteins isolation & purification, Molecular Chaperones biosynthesis, Molecular Chaperones chemistry, Molecular Chaperones genetics, Molecular Chaperones isolation & purification
- Abstract
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., (Copyright © 2019 Elsevier Inc. All rights reserved.)- Published
- 2019
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