title: Selenomethionine incorporation in proteins expressed in Lactococcus lactis.
abstract: 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 A resolution by a semiautomatic approach using selenium single-wavelength anomalous diffraction phasing.
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Ronnie P-A
authors.initials: RP
authors.lastname: Berntsson
authors.forename: Nur
authors.initials: N
authors.lastname: Alia Oktaviani
authors.forename: Fabrizia
authors.initials: F
authors.lastname: Fusetti
authors.forename: Andy-Mark W H
authors.initials: AM
authors.lastname: Thunnissen
authors.forename: Bert
authors.initials: B
authors.lastname: Poolman
authors.forename: Dirk-Jan
authors.initials: DJ
authors.lastname: Slotboom
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D013294:Lactococcus lactis / Q000235:genetics*; D008074:Lipoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012645:Selenomethionine / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D008074:Lipoproteins; D011994:Recombinant Proteins; C046506:oligopeptide-binding protein, bacteria; D012645:Selenomethionine; D000251:Adenosine Triphosphatases; C412743:OpuA protein, Lactococcus lactis