Cloning, purification and preliminary crystallographic analysis of a conserved hypothetical protein, SA0961 (YlaN), from Staphylococcus aureus

SA0961 is an unknown hypothetical protein from Staphylococcus aureus that can be identified in the Firmicutes division of Gram-positive bacteria. SA0961 was cloned and the protein was overexpressed in Escherichia coli, purified and subsequently crystallized.


Introduction
In Bacillus subtilis, ylaN encodes an open reading frame for a hypothetical protein of unknown function compromising 93 residues that has been reported to be essential for cell survival (Kobayashi et al., 2003). In Staphylococcus aureus, the YlaN homologue SA0961 consists of 91 residues with 57% sequence identity to the B. subtilis protein. Like its counterpart in B. subtilis, the sequence of SA0961 (referred to hereafter as YlaN), is particularly rich in leucine (18.7%) and lysine (11%) compared with the average composition of the set of proteins encoded in the S. aureus or B. subtilis genomes. A BLAST search with the sequence of S. aureus YlaN against the NCBI database revealed 19 sequences from a range of closely related Grampositive bacteria that belong to the Firmicutes division, a cluster of Gram-positive microbes with a low G+C content.
In the genome of both B. subtilis and S. aureus, YlaN is located upstream from ftsW, an essential gene in B. subtilis (Kobayashi et al., 2003) whose protein product is involved in cell division. However, the intergenic distance of ylaN and ftsW (about 400 bp) suggests that they are probably not part of the same operon (Wang et al., 2004).
As a contribution towards understanding the structure-function relationships of S. aureus YlaN, we have initiated the determination of its three-dimensional structure. In this paper, we describe the cloning, overexpression, purification and crystallization of YlaN and the preliminary analysis of data collected from selenomethioninecontaining (SeMet) crystals.

Cloning, overexpression and purification
The ylaN gene fragment was PCR amplified directly from genomic DNA of S. aureus strain SH1000 with the primers TTGAAAA-CGGTCGGTGAAG (forward) and TTAAAATATTAAAACT-AACATGATCCATAAC (reverse). The purified DNA fragment (273 bp) was inserted into a pETBLUE1 vector using an AccepTor vector kit (Novagen). The positive clones were confirmed by blue/ white selection and colony PCR and the extracted plasmid was transformed into Escherichia coli Tuner (DE3) (Novagen). In order to produce wild-type or SeMet-incorporated YlaN protein, the transformed E. coli Tuner strain was grown either in LB medium or in minimal medium containing 10.5 g l À1 K 2 HPO 4 , 1 g l À1 (NH 4 ) 2 PO 4 , 4.5 g l À1 KH 2 PO 4 , 0.5 g l À1 trisodium citrateÁ2H 2 O, 5 g l À1 glycerol, 0.5 g l À1 adenine, guanine, thymine and uracil, 1 mM MgSO 4 Á7H 2 O, 4 mg l À1 thiamine, 50 mg l À1 selenomethionine and 100 mg l À1 of the amino acids Lys, Phe and Thr in addition to 50 mg l À1 Ile, Leu and Val. Growth was carried out at 310 K with vigorous aeration until an OD 600 of 0.6 was attained, at which point overexpression was induced with 1 mM IPTG and growth was then continued for 5 h. The cells were harvested by centrifugation at 5000 rev min À1 for 20 min at 277 K. Analysis of the soluble fraction by SDS-PAGE showed a large overexpression band corresponding to the expected molecular weight of the protein (10 kDa).
For purification of either the wild-type or SeMet protein, cells were disrupted by sonication in 50 mM Tris-HCl pH 8.0. The cell debris and denatured proteins were removed by centrifugation at 24 500 rev min À1 for 10 min. The supernatant was collected and loaded onto a DEAE-Sepharose Fast Flow column (Amersham Biosciences) and the protein was eluted with a linear gradient of 0-0.5 M NaCl in 50 mM Tris-HCl pH 8.0. The fractions containing YlaN were combined and 4.0 M (NH 4 ) 2 SO 4 was added to give a final concentration of 1.5 M. Precipitated protein was removed by centrifugation at 24 500 rev min À1 for 10 min and the supernatant was subsequently loaded onto a column packed with Phenyl Toyo-Pearl650S (Tosoh) and eluted with a reverse gradient of (NH 4 ) 2 SO 4 from 1.5 to 0 M in 50 mM Tris-HCl pH 8.0. The fractions containing YlaN were pooled and subjected to gel-filtration chromatography using a Hi-Load Superdex 200 column (Amersham Biosciences) equilibrated with 0.5 M NaCl in 50 mM Tris-HCl pH 8.0 and eluted with the same buffer. Gel-filtration analysis shows that wild-type YlaN runs with an approximate molecular weight of 20 kDa, suggesting that the protein is predominantly a dimer in solution, although higher molecular-weight aggregates could also be observed, particularly for the SeMet protein. Peak fractions corresponding to dimeric YlaN were concentrated to 18-20 mg ml À1 in a VivaSpin concentrator (5 000 Da molecular-weight cutoff) and bufferexchanged to 10 mM Tris-HCl pH 8.0.
Approximately 6 mg pure protein was obtained from 2 l of culture, with the purity of the SeMet protein being estimated at greater than 95% as determined by SDS-PAGE. The electrospray mass spectrum of the SeMet YlaN protein suggested that the N-terminal methioninie had been cleaved and this was confirmed by conventional protein sequencing. Analysis of the molecular weight by electrospray mass spectroscopy further suggested that the selenium incorporation of the sample was greater than 90%.

Crystallization and preliminary X-ray analysis
Preliminary crystallization conditions were screened by the hanging-drop vapour-diffusion method using Hampton Research crystallization kits. Initial small cuboid crystals were observed using 0.2 M sodium acetate, 0.1 M Tris-HCl pH 8.5 and 30% PEG 4000 as the precipitant. Optimization of these conditions led to larger crystals of overall dimensions 100 Â 100 Â 100 mm from 0.2 M sodium acetate, 0.1 M Tris-HCl pH 8.5 and 20% PEG 4000. SeMet crystals with an approximate dimensions 80 Â 80 Â 80 mm were obtained under the same conditions.
For data collection, a single crystal was flash-cooled in a cryoprotectant solution consisting of 0.2 M sodium acetate, 0.1 M Tris-HCl pH 8.5 and 23% PEG 4000 and 20% glycerol at 100 K. Multiple-wavelength anomalous diffraction (MAD) data were collected from this crystal to a maximum resolution of 2.4 Å (Fig. 1) using a MAR CCD 165 detector on beamline MAD10.1 at the Daresbury Synchrotron Radiation Source (SRS). Three wavelengths were chosen near the selenium-absorption edge based on a fluorescence absorption spectrum obtained from the frozen crystal in order to maximize the f 00 component ( 1 , peak), to minimize the f 0 component ( 2 , inflection) and to maximize Áf 0 ( 3 , remote). A total 180 images, with 1 rotation per image, were collected at all three wavelengths.

Figure 1
Diffraction image from a YlaN selenomethionine crystal recorded at station 10.1 at SRS Daresbury. The crystal diffracted beyond a resolution of 2.4 Å . (Matthews, 1977). Details of the data-collection statistics are presented in Table 1. A full structure determination is under way to provide insights into the structure and possible molecular function of this protein.
This work was supported by the BBSRC. The Krebs Institute is a designated BBSRC Biomolecular Sciences Centre and a member of the North of England Structural Biology Centre. LX wishes to thank the ORS scheme and the University of Sheffield for financial support.