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 journalJournal articleResearchpeer-review

Harvard

Wang, X, Hald, H, Ernst, HA, Egebjerg, J, Christensen, KV, Gajhede, M, Kastrup, JS & Mirza, OA 2010, 'Over-expression, purification and characterization of an Asc-1 homologue from Gloeobacter violaceus', Protein Expression and Purification, vol. 71, no. 2, pp. 179-183. https://doi.org/10.1016/j.pep.2010.01.011

APA

Wang, X., Hald, H., Ernst, H. A., Egebjerg, J., Christensen, K. V., Gajhede, M., ... Mirza, O. A. (2010). Over-expression, purification and characterization of an Asc-1 homologue from Gloeobacter violaceus. Protein Expression and Purification, 71(2), 179-183. https://doi.org/10.1016/j.pep.2010.01.011

Vancouver

Wang X, Hald H, Ernst HA, Egebjerg J, Christensen KV, Gajhede M et al. Over-expression, purification and characterization of an Asc-1 homologue from Gloeobacter violaceus. Protein Expression and Purification. 2010;71(2):179-183. https://doi.org/10.1016/j.pep.2010.01.011

Author

Wang, Xiaole ; Hald, Helle ; Ernst, Heidi Asschenfeldt ; Egebjerg, Jan ; Christensen, Kenneth V ; Gajhede, Michael ; Kastrup, Jette Sandholm ; Mirza, Osman Asghar. / Over-expression, purification and characterization of an Asc-1 homologue from Gloeobacter violaceus. In: Protein Expression and Purification. 2010 ; Vol. 71, No. 2. pp. 179-183.

Bibtex

@article{5fb94a50066011df825d000ea68e967b,
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 0.2-0.4 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.",
keywords = "Former Faculty of Pharmaceutical Sciences",
author = "Xiaole Wang and Helle Hald and Ernst, {Heidi Asschenfeldt} and Jan Egebjerg and Christensen, {Kenneth V} and Michael Gajhede and Kastrup, {Jette Sandholm} and Mirza, {Osman Asghar}",
note = "Keywords: Asc-1; Amino acid transporter; Auto-induction; Bacterial homologue",
year = "2010",
doi = "10.1016/j.pep.2010.01.011",
language = "English",
volume = "71",
pages = "179--183",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press",
number = "2",

}

RIS

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