title: Three-dimensional spatial patterning of proteins in hydrogels.
abstract: The ability to create three-dimensional biochemical environments that mimic those in vivo is valuable for the elucidation of fundamental biological phenomena and pathways. To this end, we designed a system in which proteins can be photochemically patterned in three dimensions within hydrogels under physiological conditions. Fibroblast growth factor-2 (FGF2) was immobilized within agarose hydrogels that were modified with two-photon labile 6-bromo-7-hydroxycoumarin-protected thiols. Two different methods were developed for FGF2 immobilization. The first procedure relies on the protein containing free cysteines for the formation of disulfide bonds with photoexposed agarose thiols. The second procedure takes advantage of the femtomolar binding partners, human serum albumin (HSA) and albumin binding domain (ABD), which have K(D) values of ~10(-14) M. Here HSA-maleimide was chemically bound to photoexposed agarose thiols, and then the FGF2-ABD fusion protein was added to form a stable complex with the immobilized HSA. The use of orthogonal, physical binding pairs allows protein immobilization under mild conditions and can be broadly applied to any protein expressed as an ABD fusion.
authors.affiliation: Department of Chemistry, University of Toronto, 80 St George Street, Toronto, ON, Canada M5S 3H6.
authors.forename: Ryan G
authors.initials: RG
authors.lastname: Wylie
authors.forename: Molly S
authors.initials: MS
authors.lastname: Shoichet
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D040761:Biomimetic Materials / Q000032:analysis / Q000737:chemistry* / Q000494:pharmacology; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D003374:Coumarins / Q000737:chemistry; D004926:Escherichia coli; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000378:metabolism; D020100:Hydrogels / Q000737:chemistry* / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D008027:Light; D051379:Mice; D008969:Molecular Sequence Data; D058953:Neural Stem Cells / Q000187:drug effects; D055668:Photochemical Processes / Q000528:radiation effects; D017785:Photons; D010957:Plasmids; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D012685:Sepharose / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014169:Transformation, Bacterial
chemical_list: D003374:Coumarins; D020100:Hydrogels; D055767:Immobilized Proteins; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D013438:Sulfhydryl Compounds; D016222:Fibroblast Growth Factor 2; D012685:Sepharose