title: The use of a proteinaceous "cushion" with a polystyrene-binding peptide tag to control the orientation and function of a target peptide adsorbed to a hydrophilic polystyrene surface.
abstract: In immobilizing target biomolecules on a solid surface, it is essential (i) to orient the target moiety in a preferred direction and (ii) to avoid unwanted interactions of the target moiety including with the solid surface. The preferred orientation of the target moiety can be achieved by genetic conjugation of an affinity peptide tag specific to the immobilization surface. Herein, we report on a strategy for reducing the extent of direct interaction between the target moiety and surface in the immobilization of hexahistidine peptide (6His) and green fluorescent protein (GFP) on a hydrophilic polystyrene (PS) surface: Ribonuclease HII from Thermococcus kodakaraensis (cHII) was genetically inserted as a "cushion" between the PS-affinity peptide tag and target moiety. The insertion of a cushion protein resulted in a considerably stronger immobilization of target biomolecules compared to conjugation with only a PS affinity peptide tag, resulting in a substantially enhanced accessibility of the detection antibody to the target 6His peptide. The fluorescent intensity of the GFP moiety was decreased by approximately 30% as the result of fusion with cHII and the PS-affinity peptide tag but was fully retained in the immobilization on the PS surface irrespective of the increased binding force. Furthermore, the fusion of cHII did not impair the stability of the target GFP moiety. Accordingly, the use of a proteinaceous cushion appears to be promising for the immobilization of functional biomolecules on a solid surface. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:527-534, 2016.
authors.affiliation: Div. of Chemistry and Biochemistry, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Kita-Ku, Okayama, 700-8530, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Imanaka
authors.affiliation: Div. of Chemistry and Biochemistry, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Kita-Ku, Okayama, 700-8530, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Yamadzumi
authors.affiliation: Div. of Chemistry and Biochemistry, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Kita-Ku, Okayama, 700-8530, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Yanagita
authors.affiliation: Div. of Chemistry and Biochemistry, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Kita-Ku, Okayama, 700-8530, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Ishida
authors.affiliation: Div. of Chemistry and Biochemistry, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Kita-Ku, Okayama, 700-8530, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Nakanishi
authors.affiliation: Div. of Chemistry and Biochemistry, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Kita-Ku, Okayama, 700-8530, Japan.
authors.forename: Koreyoshi
authors.initials: K
authors.lastname: Imamura
mesh_terms: D000327:Adsorption; D001665:Binding Sites; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011137:Polystyrenes / Q000737:chemistry / Q000378:metabolism*; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013499:Surface Properties; D019712:Thermococcus / Q000201:enzymology
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D010455:Peptides; D011137:Polystyrenes; D049452:Green Fluorescent Proteins; D006639:Histidine; C091139:ribonuclease HII; D016914:Ribonuclease H
keywords: ELISA; affinity peptide tag; polystyrene; proteinaceous cushion; target biomolecules