title: Selective immobilization of Sonic hedgehog on benzylguanine terminated patterned self-assembled monolayers.
abstract: Patterned two-component, self-assembled monolayers on gold were produced by UV lithography. An oligo(ethylene glycol) terminated disulfide served as inert matrix reducing unspecific protein adsorption and cell adhesion. The second component of the self-assembled monolayer (SAM) presented a benzylguanine moiety for the immobilization of Sonic hedgehog (Shh) fused to a mutant O(6)-alkylguanine-DNA alkyltransferase (SNAP-tag™). The enzymatic activity of the SNAP-tag allows selective and covalent immobilization of the linked Shh. Time-of-flight secondary ion mass spectrometry verified the correct lateral distribution of the benzylguanine head groups in the patterned SAM. The quantification of unspecific and specific protein binding to mixed SAMs showed increased adsorption of albumin with increasing benzylguanine/(ethylene glycol) ratios. However, the immobilization of SNAP-tagged Shh was not blocked by pre-adsorbed albumin. Furthermore, the obtained micro-patterned substrates permitted direct immobilization of SNAP-tagged Shh even in the presence of many competing proteins from conditioned media of transfected HEK293 cells. Therefore, the presented system is suited for the controlled immobilization of fusion proteins from complex mixtures avoiding purification steps.
authors.affiliation: Karlsruhe Institute of Technology, Institute for Toxicology and Genetics, Germany.
authors.forename: Chiu-Wai
authors.initials: CW
authors.lastname: Kwok
authors.forename: Uwe
authors.initials: U
authors.lastname: Strähle
authors.forename: Yujie
authors.initials: Y
authors.lastname: Zhao
authors.forename: Tim
authors.initials: T
authors.lastname: Scharnweber
authors.forename: Simone
authors.initials: S
authors.lastname: Weigel
authors.forename: Alexander
authors.initials: A
authors.lastname: Welle
mesh_terms: D000327:Adsorption; D001672:Biocompatible Materials / Q000737:chemistry / Q000378:metabolism; D002448:Cell Adhesion; D006046:Gold / Q000737:chemistry; D006147:Guanine / Q000737:chemistry*; D057809:HEK293 Cells; D006367:HeLa Cells; D053823:Hedgehog Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008422:Materials Testing; D015394:Molecular Structure; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013499:Surface Properties
chemical_list: D001672:Biocompatible Materials; D053823:Hedgehog Proteins; D011993:Recombinant Fusion Proteins; D006147:Guanine; D006046:Gold; D019853:O(6)-Methylguanine-DNA Methyltransferase