Preliminary Study On The Application Of Inorganic Binding Peptide's Interaction With Metal Oxide Nanoparticles

Inorganic binding peptides often termed as genetically engineered polypeptides for inorganics (GEPIs) are small peptide sequences selected via combinatorial biology-based protocols of phage or cell surface display technologies. These short peptides exsit in vivo and can specifically bind to inorganics including metal and oxides etc. Recent advances in nanotechnology and molecular biology allow the genetical engineering of these peptides, used as molecular linkers or assemblers, to facilitate novel materials synthesis, which provides a new insight into the material science and engineering field. As a case study on this biomimetic application, here the functional research of inorganic binding peptides in the field of emerging interdiscipline of nanobiotechnology is reported, including its synthesis promotion in the bio-inorganic synthesis of inorganics and mediating the immobilization of inorganics on the biomolecular such as baculovirus to fabricate bio-inorganic compound structure.At first, study on the bio-inorganic interactions was conducted under the background of the prokaryotic expression system. According to the pJKS9 sequence screened by Klemm's group using a cell-surface display system, the gene encoding a Zinc Oxides-binding peptide was synthesized for a case study of protein-material interactions. It was genetically fused with His6-tag and GST-tag using E.coli expression vector pET-28a(+) and pGEX-4T-3 for the recombinant expression. The recombinant protein GST-His-ZnO was expressed, purified with NTA-Ni system, identified by SDS-PAGE electrophoresis and Western blot analysis and confirmed by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. The fusion protein GST-His-ZnO was just acquired by a facile and convenient biosynthesis method. This double-tagged bundled-up protein had a high solubility conferred by GST fusion and can be purified economically because of His tag. Results from the bio-inorganic synthesis experiment indicated that the new protein played a promoting part in grain refinement and accelerated precipitation during the formation of the ultra-fine precursor powders in the Zn(OH)2 sol solution, while the well-made intermediates are ever so vital in two-step liquid phase fabrication of ZnO nanomaterials. X-ray diffraction (XRD)analysis on the final products after calcining the precursor precipitates showed that hexagonal wurtzite ZnO crystals were obtained. The availability of the new ZnO binding protein may allow further exploration in the fields such as biosensors, biomicroarray chips, bio-inorganic material synthesis in mild aqueous environment and bioremediation of the heavy metal polluted water.In the exploratory trial of constructing organic-inorganic composites, a proof-of-principle study on combining eukaryotic baculoviral display technology with peptide-mediated immobilization of inorganic materials was conducted. The strategy was first to construct two recombinant viral vectors in which a second copy of gp64 gene and vp39 gene, respectively encoding the AcNPV(Autographa californica nuclear polyhedrosis virus) major envelope glycoprotein gp64 and major capsid protein vp39 were each introduced at the polh or p10 locus of baculovirus genome dispensable for viral propagation in vitro for the fusion with foreign genes. Then surface and capsid-modified recombinant baculovirus were respectively harvested after transfection of the identified positive recombinant bacmids into insect cells, and then led to fusion protein's expression and its display on the surface and capsid of the budded virions upon infection. Recombinant viruses were then used to fabricate bio-inorganic composites with nanoparticles. Our results showed using Bac-to-Bac expression system, His-tagged ZnO GEPI was successfully recombinated into the surface and the capsid of baculovirus respectively. Recombinant baculovirus maintained both the viral infectivity and specific binding activity of the insert protein segment. The gene construction of recombinant transfer plasmid was examined by polymerase chain reaction (PCR) analysis and enzymatic digestion identification, and verified by gene sequencing. Surface and display of the fused peptide were revealed by Western blot analysis in dissolution studies and determined by immunogold electron microscopy. Peptide-midiated adherence of the nanoparticles to the recombinant baculovirus was visualized by transmission electron microscopy (TEM) analysis. Here we demonstrated the feasibilities of combining peptide-mediated immobilization with baculovirus display technology. This work would be a valuable addition to the field of bio-inorganic composite engineering. It can facilitate the study on nanoparticles'application in biomedical detection and tracking as well as fabrication of functional complex nanodevices in the future. Base on the same stategy, Au-specific binding peptide was also engineered into the baculovirus envelope to bind to the colloid gold nanoparticles prepared according to NaHB reduction method by Tschopp et al (1982).The adsorption of colloid Au with Au-binding peptide modified recombinant baculovirus may be helpful for the development of Au-binding peptide tagged expression system, then the immunogold microscopy detection of recombinant protein can be performed more facilely and economically with the non antibody-conjugated nanogold particles.GST and His double-tagged protein and its ZnO-binding bioactivity were also reported in our work.