Part:BBa_K4428002

PbrR691

A lead-specific binding protein, PbrR, and promoter pbr from the lead resistance operon, pbr, of Cupriavidus metallidurans CH34 have shown highly sensitive and selective whole-cell detection of lead ions. In our project, we are using a recombinant version of PbrR protein, PbrR691, and displaying it on the extracellular membrane of Escherichia coli BL21 cells.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Background

While PbrR and promoter pbr from the lead resistance operon, pbr, of Cupriavidus metallidurans CH34 have shown highly sensitive and selective whole-cell detection of lead ions (Hobman et al., 2012), a PbrR homolog, PbrR691 derived from the C. metallidurans CH34 genome displays 1000 times more specificity towards lead as compared to other metal ions (P. Chen et al., 2007). Being more soluble than PbrR, PbrR691 is a more suitable choice for biosensors (P. Chen et al., 2005), however, its feasibility has not been tested yet for bioremediation applications.

In the studies where PbrR has been used for the adsorption of Pb2+ via cell surface display, its adsorption capacity has proved to be limited, indicating the requirement for improved methods. Thus, PbrR691 is used in this project with the aim of achieving enhanced adsorption capacity via cell surface display.

3D structure

The 3D structure of protein of PbrR691. From: https://www.rcsb.org/3d-sequence/5GPE?assemblyId=1

Part Uses

Recently extracellular display of proteins has become the method of choice to enhance the adsorption selectivity and the capacity of specific metal ions. This is done by anchoring specific heavy metal-binding proteins on the bacterial surface by fusing it with a naturally occurring cell surface protein (called the anchor protein).

PbrR691, a homolog of PbrR, is a soluble protein that can be easily expressed on the cell surface. On comparison of the performance of PbrR691 and PbrR, PbrR691 shows an enhanced performance (431.7‐μmol/g CDW ) compared to PbrR (388.4‐μmol/g CDW). Further, overexpression of PbrR was shown to exert a much higher burden on cell growth in comparison to PbrR691 (Jia et al.,2020).

Relative adsorption capacities for different mixed metal concentrations for PbrR, PbrR691, and PbrD (Jia et al., 2020)

The high selectivity of PbrR691 has been attributed to the unique semidirectional geometry of the lead coordination center, so that Pb²⁺ can be preferentially recognized by PbrR691 through chelation effects (Taghvi et al., 2008).