Metal-reducing enzymatic tag for optical and electron microscopy
Enzymes that reduce specific metal or metalloid ions to insoluble form are important to science. Peptides isolated from yeast- and phage-display libraries can affect the size and morphology of inorganic materials during their synthesis. Herein, an Se binding peptide was fused to an enzyme capable of reducing selenite (SeO 3 2− ) to a Se 0 nanoparticle (SeNP). The fusion of the Se binding peptide to the metalloid reductase provided size control of the resulting SeNP. The SeNP product also remains associated to the enzyme fusion. The Se binding peptide fusion to the enzyme increases the enzyme's SeO 3 2− reductase activity. Modification of enzyme activity was absent, and the size control of particles was diminished when the Se binding peptide was added exogenously to the reaction mixture. Binding of the peptide is attributed to His based ligation that results in a conformational change to the peptide.
1. A fusion protein comprising a target protein and a redox-active tag,
wherein the redox-active tag comprises:
a) a metalloid reductase that reduces a metal salt to a metal nanoparticle; and
b) a metal nanoparticle binding peptide comprising the amino acid sequence of LTPHKHHKHIHA (SEQ ID NO: 1) wherein the metal nanoparticle binding peptide binds the metal nanoparticle; and
wherein the metal nanoparticle binding peptide is fused directly to the metalloid reductase.
2. A plasmid comprising a nucleotide sequence encoding the fusion protein of claim 1 .
3. A method for detecting cloned nanoparticles in cells comprising:
a) providing cells comprising the plasmid of claim 2 and expressing the fusion protein in the cells to form tagged cells;
b) incubating the tagged cells with a metal salt to form cloned nanoparticles via enzymatic reduction of the metal salt by the metalloid reductase to form a cloned nanoparticle and retention of the cloned nanoparticle by the metal nanoparticle binding peptide; and
c) detecting the cloned nanoparticles at the target protein in the cells.
4. The method of claim 3 wherein the metalloid reductase is glutathione reductase-like metalloid reductase (GSHRMR).
5. The method of claim 4 wherein the GSHRMR reduces a salt of selenium to selenium (0) metal.
6. The method of claim 5 wherein the metal nanoparticle binding peptide binds and retains the selenium (0) via histidine moieties.
7. The method of claim 3 wherein the cloned nanoparticles have a diameter of 1 nm to 250 nm.
8. The method of claim 3 wherein a size distribution of the cloned nanoparticles is characterized by a root mean square (rms) deviation in diameter of less than 25%.
9. The method of claim 3 wherein detecting the cloned nanoparticles at the target protein in the cells is performed by scanning electron microscopy (SEM) or optical microscopy.
10. The method of claim 3 wherein the target protein is a protein from a bacterium or a virus.
11. The fusion protein of claim 1 , wherein the metal nanoparticle binding peptide consists of the amino acid sequence of LTPHKHHKHLHA (SEQ ID NO: 1).
12. The fusion protein of claim 1 , wherein the metal nanoparticle binding peptide is a selenium nanoparticle binding peptide.
13. The fusion protein of claim 1 , wherein the metal nanoparticle binding peptide is capable of binding a selenium (0) nanoparticle.
14. The fusion protein of claim 1 , wherein the metalloid reductase is GSHRMR.
15. The fusion protein of claim 1 , wherein the metalloid reductase is capable of reducing a selenium oxyanion to a selenium nanoparticle.
16. The fusion protein of claim 1 , wherein the metal salt is a selenium salt.
17. The fusion protein of claim 1 , wherein the metalloid reductase is GSHRMR and the metal nanoparticle binding peptide is a selenium nanoparticle binding peptide.
18. The fusion protein of claim 1 , wherein the metalloid reductase is GSHRMR, the metal nanoparticle binding peptide is a selenium nanoparticle binding peptide, and the metal salt is a selenium salt.
19. The fusion protein of claim 1 , wherein the metal nanoparticle is a selenium nanoparticle.
20. The fusion protein of claim 1 , wherein the metal nanoparticle has a size of 5.0±1.0 nm.
21. The fusion protein of claim 1 , wherein the metal nanoparticle is a chalcogen nanoparticle.