Lithium-Binding Peptide 3

Usage

lithium-binding peptide 3 (LBP3) is a human designed lithium-binding peptide. It can bind Li(Ⅰ) specificaly. We will display it with Pichia pastoris through cell-surface display systems.

Biology

In a study, researchers demonstrate a novel strategy for lithium extraction from battery-polluted water using a biosorbent consisting of whole cells displaying a lithium-binding peptide (LBP). For this goal, the surface of Escherichia coli was engineered to display the lithium binding peptide GPGAP (LBP) by using OmpC as an anchoring motif. Further, the lithium binding capacity of the peptide LBP was increased by construct dimers, trimers, and tetramers of LBP. The trimeric construct of LBP exhibited the highest level of lithium adsorption (3240.187 µmol g DCW− 1) at 20 mM of LiCl concentration. The trimeric construct of LBP displayed E. coli showed high lithium selectivity over cobalt, chromium, and copper respectively present in artificially wastewater^[1].

Simulation

We used the MLatom calculation program on the XACS platform to perform structural calculations of the binding between metal ion binding peptides and metal ions^[2-6], in order to predict the binding ability. In the following video: white is H, gray is C, blue is N, red is O, flesh color is Co, purple is Li, light green is Cl, dark green is Ni.
(1) Sequence
Here we perform machine learning quantum chemistry calculations for the sequence LBP3: GPGDPGPGDPGPGDP. Firstly, the geometric structure of the polypeptide chain was optimized, and the folded configuration was obtained, as shown below.
Video Embed

Vid. 1 Structure of LBP3.

(2) Structural analysis of single ion binding
We simulated the binding of a single lithium ion to a folded polypeptide chain. Through structural optimization, we get the following results:
Video Embed

Vid. 2 Structure of LBP3 binding 1 Li⁺.

It can be seen that the atoms coordinating with Li⁺ are mainly O and N atoms on the polypeptide chain. Li+ ions are encased.
(3) The combination of multiple ions
We designed this metal-binding peptide in the hope that they could trap multiple metal ions and increase efficiency. In order to analyze the binding of multiple Li⁺ ions, four Li⁺ ions were added to the molecular model, and four Cl^- ions were added to balance their charge. The total charge of the system was 0. Through structural optimization, we get the following results:
Video Embed

Vid. 3 Structure of LBP3 binding 4 Li⁺.

Li has a low coordination number and does not form clusters. It can be seen that the counterion Cl^- is less involved in coordination, and the counterion is also bound to the carbon skeleton, which can regulate the charge distribution and the structure of the polypeptide.

Experiments

1.We used Pichia Pastoris GS115 as chassis cell and pGAPZα plasmid to design the display system. By inserting the LBP3 metal-binding peptide gene as the target gene, we obtained the corresponding surface display plasmid.

Fig. 1 Electrophoretic map of plasmids containing MBPs 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.

Reference

[1]Selvamani, V., Jeong, J., Maruthamuthu, M. K., Arulsamy, K., Na, J.,... Hong, S. H. (2023). Construction of the lithium binding peptide displayed recombinant Escherichia coli for the specific lithium removal from various metal polluted wastewater. Journal of environmental chemical engineering, 11(1), 109029. doi: 10.1016/j.jece.2022.109029
[2]Sun, Q., Zhang, X., Banerjee, S., Bao, P., Barbry, M., Blunt, N.,... Chan, G. (2020). Recent developments in the PySCF program package. JOURNAL OF CHEMICAL PHYSICS, 153(2). doi: 10.1063/5.0006074
[3] Sun, Q., Berkelbach, T., Blunt, N., Booth, G., Guo, S., Li, Z.,... Chan, G. (2018). PYSCF: the Python-based simulations of chemistry framework. WILEY INTERDISCIPLINARY REVIEWS-COMPUTATIONAL MOLECULAR SCIENCE, 8(1). doi: 10.1002/wcms.1340
[4] Sun, Q. (2015). Libcint: An efficient general integral library for Gaussian basis functions. JOURNAL OF COMPUTATIONAL CHEMISTRY, 36(22), 1664-1671. doi: 10.1002/jcc.23981
[5] Wang, L., & Song, C. (2022). Geometry optimization made simple with explicit translation and rotation coordinates (vol 144, 214108, 2016). JOURNAL OF CHEMICAL PHYSICS, 157(1). doi: 10.1063/5.0102029
[6]Jmol: an open-source Java viewer for chemical structures in 3D. http://www.jmol.org/

Sequence and Features

Assembly Compatibility: