title: Completely monodisperse, highly repetitive proteins for bioconjugate capillary electrophoresis: development and characterization.
abstract: Protein-based polymers are increasingly being used in biomaterial applications because of their ease of customization and potential monodispersity. These advantages make protein polymers excellent candidates for bioanalytical applications. Here we describe improved methods for producing drag-tags for free-solution conjugate electrophoresis (FSCE). FSCE utilizes a pure, monodisperse recombinant protein, tethered end-on to a ssDNA molecule, to enable DNA size separation in aqueous buffer. FSCE also provides a highly sensitive method to evaluate the polydispersity of a protein drag-tag and thus its suitability for bioanalytical uses. This method is able to detect slight differences in drag-tag charge or mass. We have devised an improved cloning, expression, and purification strategy that enables us to generate, for the first time, a truly monodisperse 20 kDa protein polymer and a nearly monodisperse 38 kDa protein. These newly produced proteins can be used as drag-tags to enable longer read DNA sequencing by free-solution microchannel electrophoresis.
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, CA, USA.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Lin
authors.forename: Jennifer Coyne
authors.initials: JC
authors.lastname: Albrecht
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Meagher
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Wang
authors.forename: Annelise E
authors.initials: AE
authors.lastname: Barron
mesh_terms: D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D004262:DNA Restriction Enzymes / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*; D012996:Solutions
chemical_list: D009842:Oligopeptides; D011994:Recombinant Proteins; D012996:Solutions; D004247:DNA; D004262:DNA Restriction Enzymes