Selenomethionine incorporation in proteins expressed in Lactococcus lactis

Ronnie P. -A. Berntsson; Nur Alia Oktaviani; Fabrizia Fusetti; Andy-Mark W. H. Thunnissen; Bert Poolman; Dirk-Jan Slotboom

doi:10.1002/pro.97

Selenomethionine incorporation in proteins expressed in Lactococcus lactis

Ronnie P. -A. Berntsson, Nur Alia Oktaviani, Fabrizia Fusetti, Andy-Mark W. H. Thunnissen, Bert Poolman, Dirk-Jan Slotboom^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

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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 angstrom resolution by a semiautomatic approach using selenium single-wavelength anomalous diffraction phasing.

Original language	English
Pages (from-to)	1121-1127
Number of pages	7
Journal	Protein Science
Volume	18
Issue number	5
DOIs	https://doi.org/10.1002/pro.97
Publication status	Published - May-2009

Keywords

X-ray crystallography
selenomethionine incorporation
Lactococcus lactis
mass spectrometry
HUMAN CHORIONIC-GONADOTROPIN
MEMBRANE-PROTEINS
PICHIA-PASTORIS
CRYSTALLIZATION
TRANSPORT
OVERPRODUCTION
SPECIFICITY
REFINEMENT
BINDING

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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 angstrom resolution by a semiautomatic approach using selenium single-wavelength anomalous diffraction phasing.",

keywords = "X-ray crystallography, selenomethionine incorporation, Lactococcus lactis, mass spectrometry, HUMAN CHORIONIC-GONADOTROPIN, MEMBRANE-PROTEINS, PICHIA-PASTORIS, CRYSTALLIZATION, TRANSPORT, OVERPRODUCTION, SPECIFICITY, REFINEMENT, BINDING",

author = "Berntsson, {Ronnie P. -A.} and Oktaviani, {Nur Alia} and Fabrizia Fusetti and Thunnissen, {Andy-Mark W. H.} and Bert Poolman and Dirk-Jan Slotboom",

year = "2009",

month = may,

doi = "10.1002/pro.97",

language = "English",

volume = "18",

pages = "1121--1127",

journal = "Protein Science",

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T1 - Selenomethionine incorporation in proteins expressed in Lactococcus lactis

AU - Berntsson, Ronnie P. -A.

AU - Oktaviani, Nur Alia

AU - Fusetti, Fabrizia

AU - Thunnissen, Andy-Mark W. H.

AU - Poolman, Bert

AU - Slotboom, Dirk-Jan

PY - 2009/5

Y1 - 2009/5

N2 - 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 angstrom resolution by a semiautomatic approach using selenium single-wavelength anomalous diffraction phasing.

AB - 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 angstrom resolution by a semiautomatic approach using selenium single-wavelength anomalous diffraction phasing.

KW - X-ray crystallography

KW - selenomethionine incorporation

KW - Lactococcus lactis

KW - mass spectrometry

KW - HUMAN CHORIONIC-GONADOTROPIN

KW - MEMBRANE-PROTEINS

KW - PICHIA-PASTORIS

KW - CRYSTALLIZATION

KW - TRANSPORT

KW - OVERPRODUCTION

KW - SPECIFICITY

KW - REFINEMENT

KW - BINDING

U2 - 10.1002/pro.97

DO - 10.1002/pro.97

M3 - Article

SN - 0961-8368

VL - 18

SP - 1121

EP - 1127

JO - Protein Science

JF - Protein Science

IS - 5

ER -

Selenomethionine incorporation in proteins expressed in Lactococcus lactis

Abstract

Keywords

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