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.

Figure 1

Selenomethionine incorporation in OppA* and OpuA. A: Mass spectra of the tryptic peptides ALANDPEILLMDEAFSALDPLIR and VGFVVPSYMNVNSIEDLTNQANK from OppA* containing SeMet (upper panel) or methionine (lower panel). B, C: Mass spectra of intact OppA* (panel B) and OpuAA/OpuABC (panel C). Continuous lines represent methionine-containing proteins, dotted lines represent proteins with SeMet incorporated. In panel B, the triply charged peaks of OppA* are shown. In panel C, the triply charged peaks of OpuAA (m/z values of 20,984 and 21,306) and the doubly charged peaks of OpuABC (m/z 22,818 and 23,157) are shown.

Figure 2

Experimental electron density and model of OppA*. A: Stereoview of the anomalous difference Fourier map superimposed on a backbone trace of OppA*. Selenomethionine residues are highlighted in a balls-and-sticks representation (orange). The Se anomalous difference Fourier map, calculated between 61 and 2.4 Å and contoured at 5σ, is shown in blue. Thirteen of a total of 14 SeMet were visible in the map, SeMet at position 1 was in a disordered region of the protein. Peak heights are as follows: MSE-178, 36.3σ; MSE-530, 35.5σ; MSE-62, 32.7σ; MSE-45, 32.6σ; MSE-555, 22.0σ; MSE-457, 21.3σ, MSE-562, 20.2σ; MSE-448, 19.8σ, MSE-182, 17.8σ; MSE-278, 17.5σ; MSE-305, 17.3σ; MSE-300, 14.3σ; MSE-542, 11.7σ. No noise peaks were visible at a 5σ cutoff. B: Electron density (2F_o-F_c map contoured at 1.5σ) showing part of OppA*, visualizing the good quality of the experimentally derived phases. The protein is shown in a stick representation.

Figure 3

Ribbon representation of OppA* in an open unliganded conformation. The refined model contains a continuous polypeptide trace from Asn18 to Ala576. The two α/β domains are made up of residues 18–300 and 543–572 (domain I, green and blue), and residues 301–572 (domain II, orange). Domain I has two subdomains: domain I_a (residues 18–82, 220–300, and 543–573, blue) and domain I_b (residues 83–219, green). The hinge region corresponds to the segments that link domain I_a and domain II, i.e., the blue to orange transitions.