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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ATP-Binding Cassette Transporters / chemistry*
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ATP-Binding Cassette Transporters / genetics
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ATP-Binding Cassette Transporters / metabolism
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Adenosine Triphosphatases / chemistry*
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Adenosine Triphosphatases / genetics
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Adenosine Triphosphatases / metabolism
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Bacterial Proteins / chemistry*
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Carrier Proteins / chemistry*
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Carrier Proteins / genetics
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Carrier Proteins / metabolism
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Crystallography, X-Ray
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Lactococcus lactis / genetics*
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Lipoproteins / chemistry*
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Lipoproteins / genetics
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Lipoproteins / metabolism
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Mass Spectrometry
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Models, Molecular
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Recombinant Proteins / chemistry*
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Selenomethionine / metabolism*
Substances
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ATP-Binding Cassette Transporters
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Bacterial Proteins
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Carrier Proteins
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Lipoproteins
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Recombinant Proteins
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oligopeptide-binding protein, bacteria
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Selenomethionine
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Adenosine Triphosphatases
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OpuA protein, Lactococcus lactis