title: Over-expression, purification and characterization of an Asc-1 homologue from Gloeobacter violaceus.
abstract: The human alanine-serine-cysteine transporter 1 (Asc-1) belongs to the slc7a family of solute carrier transporters. Asc-1 mediates the uptake of d-serine in an exchanger-type fashion, coupling the process to the release of alanine and cysteine. Among the bacterial Asc-1 homologues, one transporter shows a significantly higher sequence identity (35%) than other bacterial homologues. Therefore, this homologue from Gloeobacter violaceus might represent the best bacterial target for structural studies probing the molecular mechanism of Asc-1. We have over-expressed the G. violaceus transporter by auto-induction, and performed purification and biophysical characterization. In addition, growth studies indicate a preference for alanine as nitrogen source in cells expressing the G. violaceus transporter. It was observed that use of the auto-induction method and subsequent optimization of the length of auto-induction was crucial for obtaining high yields and purity of the transporter. The transporter was purified with yields in the range of 0.2-0.4mg per L culture and eluted in a single peak from a size-exclusion column. The circular dichroism spectrum revealed a folded and apparently all-helical protein.
authors.affiliation: Biostructural Research, Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark.
authors.forename: Xiaole
authors.initials: X
authors.lastname: Wang
authors.forename: Helle
authors.initials: H
authors.lastname: Hald
authors.forename: Heidi A
authors.initials: HA
authors.lastname: Ernst
authors.forename: Jan
authors.initials: J
authors.lastname: Egebjerg
authors.forename: Kenneth V
authors.initials: KV
authors.lastname: Christensen
authors.forename: Michael
authors.initials: M
authors.lastname: Gajhede
authors.forename: Jette S
authors.initials: JS
authors.lastname: Kastrup
authors.forename: Osman
authors.initials: O
authors.lastname: Mirza
mesh_terms: D000409:Alanine / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000235:genetics; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary / Q000235:genetics; D012694:Serine / Q000235:genetics / Q000378:metabolism*
chemical_list: D026901:Membrane Transport Proteins; D012694:Serine; D003545:Cysteine; D000409:Alanine