Berntsson Ronnie P-A, Alia Oktaviani Nur, Fusetti Fabrizia, Thunnissen Andy-Mark W H, Poolman Bert, Slotboom Dirk-Jan
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.
Protein Sci. 2009 May;18(5):1121-7. 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 A resolution by a semiautomatic approach using selenium single-wavelength anomalous diffraction phasing.
乳酸乳球菌是用于(膜)蛋白过量表达的有前景的宿主。在此,我们描述了一种将硒代甲硫氨酸(SeMet)掺入乳酸乳球菌中表达的蛋白质的方法。通过质谱监测SeMet在来自乳酸乳球菌的膜蛋白复合物OpuA(一种ABC转运蛋白)和可溶性蛋白OppA中的掺入效率。两种蛋白质都以高效率(>90%)掺入SeMet,这极大地扩展了乳酸乳球菌作为表达宿主用于X射线晶体学目的的用途。通过使用硒单波长反常衍射相位法的半自动方法,以2.4埃分辨率测定了无配体OppA的晶体结构。