title: Combination of cysteine- and oligomerization domain-mediated protein immobilization on a surface plasmon resonance (SPR) gold chip surface.
abstract: Here we report an effective method for protein immobilization on a surface plasmon resonance (SPR) gold chip, describing the combination of cysteine- and oligomerization domain-mediated immobilization of enhanced green fluorescent protein (EGFP) as a model protein for the purpose of orientation-controlled surface density packing. In order to facilitate the oligomerization of EGFP, the dimeric and trimeric constructs derived from GCN4- leucine zipper domain were chosen for multimeric EGFP assembly. For orientation-controlled immobilization of the protein, EGFP modified with cysteine residues showing excellent orientation on a gold chip was used as a starting protein, as previously reported in our earlier study (Anal. Chem., 2007, 79, 2680-2687). Constructs of EGFP with oligomerization domains were genetically engineered, and corresponding fusion proteins were purified, applied to a gold chip, and then analyzed under SPR. The immobilized EGFP density on a gold chip increased according to the states of protein oligomerization, as dimeric and trimeric EGFPs displayed better adsorption capability than monomeric and dimeric forms, respectively. Fluorescence measurement corroborated the SPR results. Taken together, our findings indicated that the combination of cysteine- and oligomerization domain-mediated immobilization of protein could be used in SPR biosensor applications, allowing for an excellent orientation and high surface density simultaneously.
authors.affiliation: BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Eoeun-Dong, Yuseong-Gu, Daejeon, Korea.
authors.forename: Kyoungsook
authors.initials: K
authors.lastname: Park
authors.forename: Jeong Min
authors.initials: JM
authors.lastname: Lee
authors.forename: Yongwon
authors.initials: Y
authors.lastname: Jung
authors.forename: Tsegaye
authors.initials: T
authors.lastname: Habtemariam
authors.forename: Abdela Woubit
authors.initials: AW
authors.lastname: Salah
authors.forename: Cesar D
authors.initials: CD
authors.lastname: Fermin
authors.forename: Moonil
authors.initials: M
authors.lastname: Kim
mesh_terms: D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods; D003545:Cysteine / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D055767:Immobilized Proteins / Q000737:chemistry; D016350:Leucine Zippers; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; C501391:GCN4 protein, S cerevisiae; D055767:Immobilized Proteins; D029701:Saccharomyces cerevisiae Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006046:Gold; D003545:Cysteine