title: Profiling of membrane protein variants in a baculovirus system by coupling cell-surface detection with small-scale parallel expression.
abstract: Production of structure-grade mammalian membrane proteins in substantial quantities has been hindered by a lack of methods for effectively profiling multiple constructs expression in higher eukaryotic systems such as insect or mammalian cells. To address this problem, a specialized small-scale eukaryotic expression platform by Thomson Instrument Company (Vertiga-IM) was developed and used in tandem with a Guava EasyCyte microcapillary 96-well cytometer to monitor cell density and health and evaluate membrane protein expression. Two proof of concept experiments were conducted using the human beta(2)-adrenergic receptor (beta(2)AR) and the gap junction protein connexin26 (Cx26) in a baculovirus expression system. First, cell surface expression was used to assess the expression levels of 14 beta(2)AR truncation variants expressed using the Vertiga-IM shaker. Three of these variants were then compared to wild-type beta(2)AR using three metrics: cell surface expression, saturation ligand binding and protein immunoblot analysis of dodecylmaltoside extracted material. Second, a series of systematic Cx26 truncation variants were evaluated for expression by protein immunoblot analysis. The cumulative results for these two systems show that the Vertiga-IM instrument can be used effectively in the parallel insect cell microexpression of membrane protein variants, and that the expression of cell surface molecules as monitored with the Guava EasyCyte instrument can be used to rapidly assess the production of properly folded proteins in the baculovirus expression system. This approach expedites the in vitro evaluation of a large number of mammalian membrane protein variants.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Hanson
authors.forename: Alexei
authors.initials: A
authors.lastname: Brooun
authors.forename: Kent A
authors.initials: KA
authors.lastname: Baker
authors.forename: Veli-Pekka
authors.initials: VP
authors.lastname: Jaakola
authors.forename: Chris
authors.initials: C
authors.lastname: Roth
authors.forename: Ellen Y T
authors.initials: EY
authors.lastname: Chien
authors.forename: Alexander
authors.initials: A
authors.lastname: Alexandrov
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Velasquez
authors.forename: Leila
authors.initials: L
authors.lastname: Davis
authors.forename: Mark
authors.initials: M
authors.lastname: Griffith
authors.forename: Kin
authors.initials: K
authors.lastname: Moy
authors.forename: Barbie K
authors.initials: BK
authors.lastname: Ganser-Pornillos
authors.forename: Yuanzi
authors.initials: Y
authors.lastname: Hua
authors.forename: Peter
authors.initials: P
authors.lastname: Kuhn
authors.forename: Sam
authors.initials: S
authors.lastname: Ellis
authors.forename: Mark
authors.initials: M
authors.lastname: Yeager
authors.forename: Raymond C
authors.initials: RC
authors.lastname: Stevens
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism*; D015153:Blotting, Western; D000072259:Connexin 26; D017630:Connexins / Q000096:biosynthesis* / Q000235:genetics; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis*; D018343:Receptors, Adrenergic, beta-2 / Q000096:biosynthesis* / Q000235:genetics; D018411:Spodoptera
chemical_list: D017630:Connexins; C000607042:GJB2 protein, human; D008565:Membrane Proteins; D018343:Receptors, Adrenergic, beta-2; D000072259:Connexin 26