Selenomethionine incorporation in proteins expressed in Lactococcus lactis

• Published in: Protein science Vol. 18; no. 5; pp. 1121 - 1127
• Main Authors: Berntsson, Ronnie P.‐A., Alia Oktaviani, Nur, Fusetti, Fabrizia, Thunnissen, Andy‐Mark W. H., Poolman, Bert, Slotboom, Dirk‐Jan
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-05-2009
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; ATP-Binding Cassette Transporters - chemistry; ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette Transporters - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Crystallography, X-Ray; For the Record; Lactococcus lactis; Lactococcus lactis - genetics; Lipoproteins - chemistry; Lipoproteins - genetics; Lipoproteins - metabolism; Mass Spectrometry; Models, Molecular; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Selenomethionine - metabolism; selenomethionine incorporation; X‐ray crystallography
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.97

Lactococcus lactis is a promising host for (membrane) protein overproduction. Here, we describe a protocol for incorporation of selenomethionine (SeMet) into proteins expressed in L. lactis. Incorporation efficiencies of SeMet in the membrane protein complex OpuA (an ABC transporter) and the soluble protein OppA, both from L. lactis, were monitored by mass spectrometry. Both proteins incorporated SeMet with high efficiencies (>90%), which greatly extends the usefulness of the expression host L. lactis for X‐ray crystallography purposes. The crystal structure of ligand‐free OppA was determined at 2.4 Å resolution by a semiautomatic approach using selenium single‐wavelength anomalous diffraction phasing.