Part:BBa_K5166009

BH2807

Usage

BH2807 is a manganese-binding protein from Bacillus halodurans. It can bind Mn(Ⅱ) specificaly. We will display it with Pichia pastoris through cell-surface display systems.

Biology

The MntR homologue (BH2807, BhMntR) in Bacillus halodurans is a protein consisting of 139 amino acids, and has 78% sequence identity with MntR from B.subtilis (BsMntR). BhMntR is composed of two distinct domains in the homodimeric form, and its overall structure is similar to those of other MntR homologues. The two manganese ions formed a binuclear cluster in the metal binding site of BhMntR, via six amino acid residues; three strictly conserved residues (His77, Glu102 and His103) in the IdeR/MntR family, two residues (Asp8 and Glu99) conserved in the MntR family, and a Glu11 conserved in MntR from B. subtilis and E. coli. The manganese ion in A site was liganded with heptageometry as shown in BsMntR, whereas the manganese ion in the C site was incompletely liganded with five atoms. The sixth atom, the carbonyl oxygen of Glu102, was too far away to coordinate with the MnC ion. Therefore, BhMntR did not cause movement of the domain to bind DNA upon manganese ion binding. Binuclear metal ions were not formed in the other subunit due to the crystal packing and the flipping of His77. The side chain of His77 was flipped and stabilized by hydrogen bonding and hydrophobic stacking. In order to initiate metal binding, the side chain of His77 was flipped to interact with the carboxylate of Glu81. Although the functional assignment of metal binding site for BhMntR is tentative, this structural model is applicable to other MntR homologous structures^[1].

Experiments

1.We used Pichia Pastoris GS115 as chassis cell and pGAPZα plasmid to design the display system. By inserting the BH2807 metal-binding peptide gene as the target gene, we obtained the corresponding surface display plasmid.

Fig. 1 Electrophoretic map of plasmids containing Metal-binding peptides gene.

2.After constructing the plasmid, we introduced it into Escherichia coli for amplification. After amplification, the plasmids were extracted and purified, and sent for sequencing. After obtaining the correct sequencing results, the plasmid was transformed into Pichia pastoris by means of electrical stimulation. Finally, by colony PCR, we determined that the plasmid was successfully introduced into the yeast.

Fig. 2 Electrophoretic map of engineered yeast after colony PCR.

3.After obtaining the engineered yeast, we designed some experimental schemes to qualitatively test their adsorption effect on target metal ions. In order to test the adsorption effect, the engineered yeast and the prepared single metal solution were mixed according to a certain proportion, removed and centrifuged after 2 hours, and the supernatant and precipitation were stored respectively. We chose to use a graphite furnace to detect the concentration of metal ions in the supernatant. By calculating the ratio of the reduced metal concentration in the supernatant to the original added metal concentration, we obtained the adsorption rate of the engineered strain on the target metal ions, and made a comparison to select the strain with better adsorption effect.

Fig. 3 The adsorption rate of mntR-pir, TssS-pir and BH2807-pir for Mn²⁺ within 2 hours.

4.The cell precipitation after adsorption was used for electron microscopy sample preparation. Yeast cells with metal binding peptides on their surfaces were observed by scanning electron microscopy. Figure (a) and (b) are the control group GS115; Figures (c) and (d) show the strains of BH2807 on the surface. Compared with the photos, it was found that the roughness of cell surface was from large to small: BH2807-pir > GS115. Through literature research, we believe that the roughness of cell surface is related to the amount of displayed proteins, and the more display proteins, the rougher the cell surface. This also confirms the comparison of adsorption effect of engineered strains to a certain extent

Fig. 4 Scanning electron microscope images of yeast strains. a) GS115 (×25000 times); b) GS115 (×40000 times);c) BH2807-pir(×25000 times); d) BH2807-pir (×40000 times).

Reference

[1]Lee, M. Y., Lee, D. W., Joo, H. K., Jeong, K. H., Lee, J. Y.,... Permyakov, E. A. (2019). Structural analysis of the manganese transport regulator MntR from Bacillus halodurans in apo and manganese bound forms. PloS one, 14(11), e224689. doi: 10.1371/journal.pone.0224689.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 373
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 373
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 373
  
• 1000
  COMPATIBLE WITH RFC[1000]