Expression, purification, crystallization and crystallographic study of Lutzomyia longipalpis LJL143

LJL143, a salivary protein from L. longipalpis, was produced using P. pastoris and crystallized in space group P212121.


Introduction
Leishmaniasis is a neglected tropical disease that is endemic in over 88 countries and is caused by over 20 species and subspecies of parasitic protozoa of the genus Leishmania. Clinical manifestations of leishmaniasis vary from self-healing skin lesions to fever, anemia, skin destruction and death (Pavli & Maltezou, 2010). There are four main clinical forms of leishmaniasis: visceral leishmaniasis, cutaneous leishmaniasis, mucocutaneous leishmaniasis and diffuse cutaneous leishmaniasis. Visceral leishmaniasis is the most serious form and can be fatal if left untreated. While most cases of cutaneous leishmaniasis are mild and heal without treatment, other forms of leishmaniasis are treated with pentavalent antimony drugs, including meglumine antimonate and sodium stibogluconate (Mohamed-Ahmed et al., 2012;Maltezou, 2008). Drug resistance has been reported, leading to the use of more toxic and less effective compounds such as amphotericin B (Mohamed-Ahmed et al., 2012;Maltezou, 2008). Alternative control strategies are needed because of the failure of current therapeutics and the presence of many reservoir hosts (http:// www.who.int/tdr/diseases/leish/info/en/index.html).
Leishmania parasites are transmitted to humans by sandflies of the genus Phlebotomus and Lutzomyia in the Old and New Worlds, respectively (Collin et al., 2009). To facilitate its blood meal, a sandfly injects saliva into the host, which enhances the survival of injected Leishmania promastigotes and the establishment of disease transmission (Titus & Ribeiro, 1988;Valenzuela et al., 2001;Bezerra & Teixeira, 2001;Andrade et al., 2007;Gomes et al., 2008;Costa et al., 2013). Additionally, the sandfly salivary proteins result in the ISSN 2053-230X production of antibodies that can serve as markers of exposure (Belkaid et al., 1998). LJL143 from the saliva of the New World sandfly L. longipalpis is a known marker of exposure in zoonotic reservoirs (Teixeira et al., 2010;Collin et al., 2009). Structural studies of LJL143 were initiated as part of ongoing efforts to characterize the structure and functions of sandfly antigens. The production, crystallization and preliminary X-ray diffraction studies of LJL143 are presented.

Materials and methods
2.1. Macromolecule production DNA coding for the mature LJL143 peptide and a C-terminal hexahistidine tag was codon-optimized based on the yeast codon preference (GenScript, Piscataway, New Jersey, USA). The synthesized gene included EcoRI and XbaI restriction sites for subcloning into the pPICZ A vector ( Table 1). The plasmid DNA was linearized and transformed into Pichia pastoris strain X33 by electroporation as described previously (Zhan et al., 2005). 20 colonies with the correct insert were screened for the expression of recombinant LJL143 protein with 0.5% methanol at 303 K for 72 h. The highest expressing colony was used to make a 1 l starter culture using buffered minimal glycerol medium (BMGY; 100 mM potassium phosphate pH 6.0, 1.34% yeast nitrogen base, 4 Â 10 À5 % biotin, 1% yeast extract, 1% glycerol). Approximately 250 ml of the starter culture was added to a 14 l fermentation vessel containing 5 l basal salt medium (BSM; 26.7 ml 85% phosphoric acid, 0.93 g calcium sulfate, 18.2 g potassium sulfate, 14.9 g magnesium sulfate, 4.13 g potassium hydroxide and 40 g glycerol per litre). The BSM was maintained at pH 5 by the addition of 14% ammonium hydroxide. Antifoam 204 was added to minimize foaming. About 16 h into fermentation a dissolved-oxygen (DO) spike from the depletion of glycerol was observed, which prompted the initiation of a fed-batch phase. During the fed-batch phase, 50% glycerol was added to the culture at 15 ml per litre per hour for 6 h. In the last 2 h of the fed-batch phase, the pH of the culture was ramped from 1 to 6.0 and the temperature was reduced by 2 K. The fed-batch phase was followed by an 8 h methanol-adaptation phase during which the agitation was increased to 700 rev min À1 and a methanol feed was ramped to 11 ml per litre per hour, which was maintained until the end of fermentation.
After 72 h of induction, the culture medium containing the secreted LJL143 protein was separated from the yeast cells by centrifugation at 12 000g for 30 min and filtered with a 0.22 mm membrane filter. The clarified supernatant was bufferexchanged into the binding buffer (500 mM sodium chloride, 5 mM imidazole, 20 mM Tris pH 8.0) using a 3 kDa hollowfiber cartridge (GE Healthcare). LJL143 was purified by immobilized metal-ion affinity chromatography (IMAC) using a 5 ml HisTrap FF column (GE Healthcare). Nonspecifically bound proteins were removed by sequential steps of washing with 5, 10, 25 and 55 mM imidazole prior to protein elution with 500 mM imidazole. LJL143 was purified to $99% as assessed by Coomassie-stained SDS-PAGE (Fig. 1a)    Composition of reservoir solution 0.1 M bis-tris pH 6.5, 28% PEG 2000 MME Volume and ratio of drop 5.5 ml, 4.5:1 Volume of reservoir (ml) 500 using both anti-His-tag monoclonal antibody (Fig. 1b) and mouse antiserum against LJL143 (Fig. 1c).

Crystallization
Recombinant LJL143 was buffer-exchanged and concentrated to 23 mg ml À1 in 50 mM sodium citrate buffer pH 5.0 using a 10K cutoff centrifugal concentrating device (Millipore). The protein concentration was measured by the absorbance at 280 nm prior to setting up crystallization experiments.
Initial crystallization screens were carried out using commercial sparse-matrix screens from Hampton Research (Crystal Screen, Crystal Screen 2 and Index) at 298 K by vapour diffusion in sitting drops (Table 2).

Data collection and processing
The crystals were transferred for $30 s into cryoprotecting solution (0.1 M bis-tris pH 6.5, 28% PEG 2000 MME, 15% glycerol) and were flash-cooled directly in a stream of N 2 gas at 113 K prior to collecting diffraction data. X-ray diffraction data were collected at the Baylor College of Medicine core facility using a Rigaku HTC detector (Table 3). The X-ray source was a Rigaku FR-E+ SuperBright microfocus rotatinganode generator with VariMax HF optics. A data set was collected from a single crystal using the CrystalClear (d*TREK) package (Pflugrath, 1999) and was processed using MOSFLM (Leslie, 2006).

Results and discussion
The typical yield of LJL143 was 50 mg per litre of fermentation supernatant. The resulting protein had an electrophoretic mobility of 42 kDa. The predicted theoretical molecular mass of LJL143 is 33 594.92 Da. LJL143 has two predicted N-glycosylation sites at 42 NQTH and 241 NKTC, which may contribute to the almost 10 kDa increase in molecular mass. LJL143 crystals were grown using different polyethylene glycols (PEG) as precipitants. The best diffracting crystals were obtained after 48 h in the conditions detailed in Table 2. These crystals are illustrated in Fig. 2(a). Diffraction images reveal visible diffraction spots beyond 2.6 Å resolution (Fig. 2b). The crystal belonged to the orthorhombic space group P2 1 2 1 2 1 , with approximate unit-cell parameters a = 57.5, b = 70.2, c = 79.5 Å (Table 3). Based on the estimated Matthews coefficient and solvent-content prediction (http:// www.ruppweb.org/Mattprob), a monomer is expected in the asymmetric unit (Matthews, 1968;Kantardjieff & Rupp, 2003    This corresponds to a unit-cell volume of 321 104 Å 3 (with an asymmetric unit of 80 276 Å 3 ), a Matthews coefficient of 2.39 Å 3 Da À1 and an estimated solvent content of 48.5%. A BLAST analysis of LJL143 against the Protein Data Bank revealed no suitable homologues for molecular replacement. The closest structural homologue, 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase from Thermus thermophilus (PDB entry 2dfu; RIKEN Structural Genomics/Proteomics Initiative, unpublished work), shares only 21% sequence identity over a 26% query coverage. There are only ten S atoms out of a total of 4668 atoms, and our attempts at phase determination by single-wavelength anomalous dispersion using S atoms at the Cu K wavelength failed. Future studies will include other methods of experimental phase determination such as multi-wavelength anomalous dispersion phasing with selenomethionine-derivatized protein and heavyatom derivatization for multiple isomorphous replacement (Ingram et al., 1956;Perutz, 1953;Harker, 1956;Hendrickson & Ogata, 1997). LJL143 and other salivary-gland proteins play key roles in the transmission of leishmaniasis. The structure of LJL143 will give key insights into the underlying mechanisms of this poorly characterized family of proteins.