Recombinant expression and purification of a functional bacterial metallo-chaperone PbrD-fusion construct as a potential biosorbent for Pb(II)

Keshav V; Achilonu I; Dirr Hw; Kondiah K

doi:10.1016/j.pep.2019.02.008

Recombinant expression and purification of a functional bacterial metallo-chaperone PbrD-fusion construct as a potential biosorbent for Pb(II)

Protein Expr Purif. 2019 Jun:158:27-35. doi: 10.1016/j.pep.2019.02.008. Epub 2019 Feb 15.

Authors

Keshav V¹, Achilonu I², Dirr Hw², Kondiah K³

Affiliations

¹ School of Molecular and Cell Biology, University of Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa. Electronic address: kulsumk@uj.ac.za.
² School of Molecular and Cell Biology, University of Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa.
³ School of Molecular and Cell Biology, University of Witwatersrand, Private Bag 3, Wits, 2050, Johannesburg, South Africa; Department of Biotechnology and Food Technology, University of Johannesburg, P.O. Box 17011, Doornfontein, 2028, Johannesburg, South Africa.

PMID: 30776426
DOI: 10.1016/j.pep.2019.02.008

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.

Keywords: Biosorption; Metallo-chaperone; Pb(II); PbrD protein.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cupriavidus / chemistry*
Cupriavidus / genetics
Lead / chemistry*
Metalloproteins* / biosynthesis
Metalloproteins* / chemistry
Metalloproteins* / genetics
Metalloproteins* / isolation & purification
Molecular Chaperones* / biosynthesis
Molecular Chaperones* / chemistry
Molecular Chaperones* / genetics
Molecular Chaperones* / isolation & purification
Recombinant Proteins / biosynthesis
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / isolation & purification

Substances

Metalloproteins
Molecular Chaperones
Recombinant Proteins
Lead

Supplementary concepts

Cupriavidus metallidurans