Over-expression, purification and characterization of an Asc-1 homologue from Gloeobacter violaceus

Research output: Contribution to journalJournal articleResearchpeer-review

Xiaole Wang, Helle Hald, Heidi Asschenfeldt Ernst, Jan Egebjerg, Kenneth V Christensen, Michael Gajhede, Jette Sandholm Kastrup, Osman Asghar Mirza

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 0.2-0.4 mg 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.
Original languageEnglish
JournalProtein Expression and Purification
Volume71
Issue number2
Pages (from-to)179-183
ISSN1046-5928
DOIs
Publication statusPublished - 2010

Bibliographical note

Keywords: Asc-1; Amino acid transporter; Auto-induction; Bacterial homologue

ID: 17114872