Over-expression, purification and characterization of an Asc-1 homologue from Gloeobacter violaceus.
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.
Wang
Xiaole
X
Biostructural Research, Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, Copenhagen, Denmark.
Hald
Helle
H
Ernst
Heidi A
HA
Egebjerg
Jan
J
Christensen
Kenneth V
KV
Gajhede
Michael
M
Kastrup
Jette S
JS
Mirza
Osman
O
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*
D026901:Membrane Transport Proteins; D012694:Serine; D003545:Cysteine; D000409:Alanine