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Characterization of Recombinant His-Tag Protein Immobilized onto Functionalized Gold Nanoparticles.

ABSTRACT: The recombinant polyhistidine-tagged hemoglobin I ((His)?-rHbI) from the bivalve Lucina pectinata is an ideal biocomponent for a hydrogen sulfide (H?S) biosensor due to its high affinity for H?S. In this work, we immobilized (His)?-rHbI over a surface modified with gold nanoparticles functionalized with 3-mercaptopropionic acid complexed with nickel ion. The attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) analysis of the modified-gold electrode displays amide I and amide II bands characteristic of a primarily ?-helix structure verifying the presence of (His)?-rHbI on the electrode surface. Also, X-ray photoelectron spectroscopy (XPS) results show a new peak after protein interaction corresponding to nitrogen and a calculated overlayer thickness of 5.3 nm. The functionality of the immobilized hemoprotein was established by direct current potential amperometry, using H?S as the analyte, validating its activity after immobilization. The current response to H?S concentrations was monitored over time giving a linear relationship from 30 to 700 nM with a corresponding sensitivity of 3.22 × 10-3 nA/nM. These results confirm that the analyzed gold nanostructured platform provides an efficient and strong link for polyhistidine-tag protein immobilization over gold and glassy carbon surfaces for a future biosensors development.

SUBMITTER: Torres-Gonzalez L

PROVIDER: S-EPMC6308469 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Characterization of Recombinant His-Tag Protein Immobilized onto Functionalized Gold Nanoparticles.

Torres-González Lisa L Díaz-Ayala Ramonita R Vega-Olivencia Carmen A CA López-Garriga Juan J

Sensors (Basel, Switzerland) 20181204 12

The recombinant polyhistidine-tagged hemoglobin I ((His)₆-rHbI) from the bivalve <i>Lucina pectinata</i> is an ideal biocomponent for a hydrogen sulfide (H₂S) biosensor due to its high affinity for H₂S. In this work, we immobilized (His)₆-rHbI over a surface modified with gold nanoparticles functionalized with 3-mercaptopropionic acid complexed with nickel ion. The attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) analysis of the modified-gold electrode displays amid ...[more]

PMID: 30518079

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Project description:Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with "tags" that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond.

Dataset Information

Characterization of Recombinant His-Tag Protein Immobilized onto Functionalized Gold Nanoparticles.

Publications

Characterization of Recombinant His-Tag Protein Immobilized onto Functionalized Gold Nanoparticles.

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