Over-expression, purification and characterization of an Asc-1 homologue from Gloeobacter violaceus
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Over-expression, purification and characterization of an Asc-1 homologue from Gloeobacter violaceus. / Wang, Xiaole; Hald, Helle; Ernst, Heidi Asschenfeldt; Egebjerg, Jan; Christensen, Kenneth V; Gajhede, Michael; Kastrup, Jette Sandholm; Mirza, Osman Asghar.
In: Protein Expression and Purification, Vol. 71, No. 2, 2010, p. 179-183.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Over-expression, purification and characterization of an Asc-1 homologue from Gloeobacter violaceus
AU - Wang, Xiaole
AU - Hald, Helle
AU - Ernst, Heidi Asschenfeldt
AU - Egebjerg, Jan
AU - Christensen, Kenneth V
AU - Gajhede, Michael
AU - Kastrup, Jette Sandholm
AU - Mirza, Osman Asghar
N1 - Keywords: Asc-1; Amino acid transporter; Auto-induction; Bacterial homologue
PY - 2010
Y1 - 2010
N2 - 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.
AB - 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.
KW - Former Faculty of Pharmaceutical Sciences
U2 - 10.1016/j.pep.2010.01.011
DO - 10.1016/j.pep.2010.01.011
M3 - Journal article
C2 - 20074644
VL - 71
SP - 179
EP - 183
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
IS - 2
ER -
ID: 17114872