Difference between revisions of "Part:BBa K3945001"
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<p> We have successfully characterized lanmodulin’s REE binding capabilities. To do this we conducted a metal recovery assay where an initial sample of neodymium was incubated with lanmodulin. In this assay an initial known sample of neodymium was incubated with the protein. The protein fraction was then removed using centrifugal filtration and the filtrate was analyzed using the colorimetric Arsenazo assay. </p> | <p> We have successfully characterized lanmodulin’s REE binding capabilities. To do this we conducted a metal recovery assay where an initial sample of neodymium was incubated with lanmodulin. In this assay an initial known sample of neodymium was incubated with the protein. The protein fraction was then removed using centrifugal filtration and the filtrate was analyzed using the colorimetric Arsenazo assay. </p> | ||
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<p>Results from the Arsenazo was quantified by measuring absorbance at 650 nm. It was shown that the concentration of neodymium in the sample dropped from an initial value of 146 uM to 28 uM after incubation with lanmodulin, indicating an 81% neodymium recovery rate. Whereas the GST (negative control) left more than half of the neodymium in the solution, indicating that the observed neodymium binding capacity is unique to lanmodulin. </p> | <p>Results from the Arsenazo was quantified by measuring absorbance at 650 nm. It was shown that the concentration of neodymium in the sample dropped from an initial value of 146 uM to 28 uM after incubation with lanmodulin, indicating an 81% neodymium recovery rate. Whereas the GST (negative control) left more than half of the neodymium in the solution, indicating that the observed neodymium binding capacity is unique to lanmodulin. </p> | ||
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===References=== | ===References=== | ||
Latest revision as of 21:56, 20 November 2021
Lanmodulin with a C-terminus 6x His-Tag (LanM-His)
Usage and Biology
Lanmodulin is recently a novel lanthanide binding protein from Methylobacterium extorquens that displays more than 100-million fold selectivity for rare earth elements [1]. Such affinity and selectivity have not been observed in any previously studied macromolecule. In addition, lanmodulin is extremely robust, capable of withstanding temperatures as high as 95 °C and pH levels as low as 2.5 [2]. Thus, allowing it to be the perfect molecular tool for us in an efficient rare earth recovery system.
Design
The lanmodulin sequence has been codon-optimized for expression in E. coli and with the addition of a 6x histidine tag to the C-terminus of the protein, it could easily be purified using the universal His-Tag purification protocol.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 88
- 1000COMPATIBLE WITH RFC[1000]
Characterization
We have successfully characterized lanmodulin’s REE binding capabilities. To do this we conducted a metal recovery assay where an initial sample of neodymium was incubated with lanmodulin. In this assay an initial known sample of neodymium was incubated with the protein. The protein fraction was then removed using centrifugal filtration and the filtrate was analyzed using the colorimetric Arsenazo assay.
A darker color represents a high neodymium concentration while a lighter color represents a low neodymium concentration. After the addition of lanmodulin, the solution turned from a dark blue color to a light pink color. Thus, suggesting that lanmodulin was able to grab onto most of the neodymium.
Results from the Arsenazo was quantified by measuring absorbance at 650 nm. It was shown that the concentration of neodymium in the sample dropped from an initial value of 146 uM to 28 uM after incubation with lanmodulin, indicating an 81% neodymium recovery rate. Whereas the GST (negative control) left more than half of the neodymium in the solution, indicating that the observed neodymium binding capacity is unique to lanmodulin.
References
1. JA C, ER F, JA M, JV H, TN L. Lanmodulin: A Highly Selective Lanthanide-Binding Protein from a Lanthanide-Utilizing Bacterium. Journal of the American Chemical Society. 2018 [accessed 2021 Sep 17];140(44):15056–15061. https://pubmed.ncbi.nlm.nih.gov/30351021/. doi:10.1021/JACS.8B09842
2. GJ D, JA M, DM P, DW R, JA C, Y J. Selective and Efficient Biomacromolecular Extraction of Rare-Earth Elements using Lanmodulin. Inorganic chemistry. 2020 [accessed 2021 Sep 17];59(17):11855–11867. https://pubmed.ncbi.nlm.nih.gov/32686425/. doi:10.1021/ACS.INORGCHEM.0C01303