title: Targeting the expression of functional murine CMP-sialic acid transporter to the E. coli inner membrane.
abstract: The heterologous expression of functional mammalian integral membrane proteins still represents a significant hurdle towards their crystallization and structure elucidation. We have therefore explored the use of the OmpA signal sequence to deliberately target the expression of the murine CMP-sialic acid transporter, a Golgi-resident protein with 10 putative transmembrane domains, to the Escherichia coli inner membrane. Here, we show that the expression of an OmpA signal sequence-FLAG-CMP-sialic acid transporter fusion protein in E. coli results in the targeting and insertion of recombinant protein within the inner membrane. Significantly, functionality was confirmed by the ability of spheroplasted E. coli and mixed phosphatidylcholine-E. coli inner membrane proteoliposomes incorporating recombinant CMP-sialic acid transporter to accumulate CMP-sialic acid in vitro.
authors.affiliation: Institute for Glycomics, Gold Coast Campus Griffith University, Queensland 4222, Australia.
authors.forename: Andrea
authors.initials: A
authors.lastname: Maggioni
authors.forename: Mark
authors.initials: M
authors.lastname: von Itzstein
authors.forename: Rita
authors.initials: R
authors.lastname: Gerardy-Schahn
authors.forename: Joe
authors.initials: J
authors.lastname: Tiralongo
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005821:Genetic Techniques; D006056:Golgi Apparatus / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012794:Sialic Acids / Q000378:metabolism; D013104:Spheroplasts / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D011994:Recombinant Proteins; D012794:Sialic Acids; C078167:OMPA outer membrane proteins; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D019158:N-Acetylneuraminic Acid