BamH I restriction sites of the modified pET17 expression vector, pETnef, similar to mouse a-LA as described earlier [27]. The hLA-ext and hLA with N-terminal Histidine-tag (His-hLA) were constructed in Nde I and EcoR I restriction sites of the pETnef vector, similar to the glutathione S-transferase protein with the same C-terminal extension, as described previously [26]; the deletion of the b-domain in the human a-LA, the aD-hLA, was constructed as described previously [28]; the construction of aD- hLA with C-terminal extension (aD-hLA-ext) was similar to the previously published method [26]. From 1 L of bacterial culture, 15 mg of aD-hLA, and 5.32 mg of aD-hLA-ext were obtained. All the clones were sequenced and transfected into BL21 DE3LysS cells for protein expression. The expression and refolding of these proteins were carried out under conditions similar to those described previously for mouse a-LA [27,28]. Nearly 5 mgs and 6 mgs, of purified protein were obtained from 1 L of bacterial culture for hLA and hLA-ext, respectively. For His- hLA purification, after folding protein was dialyzed first against 10 mM Tris-HCl (pH 8.0) and then against PBS, and next purified using TALON metal affinity resin (Clontech). Protein was eluted from the column in PBS buffer containing 1 M NaCl and 100 mM imidazol (pH 7.4). Elution buffer was removed by dialysis against 20 mM Tris-HCl (pH 8.0). Confirmation of the C-terminal extension with thrombin cleavage Seventeen micrograms of purified hLA-ext was incubated overnight at room temperature with one unit of thrombin from human plasma from Sigma-Aldrich (St Louis, MO) in a thrombin reaction buffer (10 mM Tris-HCl (pH 8.0), 2 mM CaCl2, and 150 mM NaCl). Equal amounts of cut and uncut proteins were analyzed by 18% SDS-PAGE and by mass spectrometry. Circular dichroism spectroscopy Near (320 to 250 nm) UV circular dichroism spectroscopy (CD) spectra were measured with AVIV Mod.202 CD Spectrometer Aviv Instruments (Lakewood, NJ) with a 10-mm light path CD UV Hellma quartz cell (Plainview, NY). The protein concentra- tion was measured by absorbance at 280 nm. All measurements were made at 25oC. The wavelength step was 1 nm, with response time 5 sec; one scan was performed, and the waiting time was 0.5 sec. The spectrum of the pure buffer was subtracted from the protein spectra. The molar ellipticity T (mdeg x cm2 x dmol21) was calculated from milidegree, the number of amino acids (aa), protein concentration (c, in molar units), and cell length (l, in cm) as T = milidegrees/ (aa x c x l). a-Lactalbumin conjugation and labeling Recombinant ppGalNAcT2 enzyme was prepared as previously described [5].Forty mg of hLA-ext was glycosylated with 20 mg of ppGalNAc-T2 overnight at room temperature in the presence of 25 mM Tris-HCl (pH 8.0), 10 mM MnCl2, and 0.5 mM UDP- C2-keto-Gal in a total volume of 100 mL. To label hLA-ext with the fluorescent probe, 7 mL of 10 mg/ml C2-keto-Gal glycosylated hLA-ext protein were labeled in a 40-mL volume containing 166 mM sodium acetate, pH 4.9, and 0.675 mg/ml aminooxy- Alexa Fluor 488 (Invitrogen, Carlsbad, CA). The reaction mixture was incubated overnight in the dark, at room temperature, in a rocking platform. Excess Alexa Fluor 488 was removed by ammonium sulfate precipitation of the labeled protein. Excess ammonium sulfate was removed by washing with 20 mM Tris (pH 8) in 10,000 MWCO centrifugal filters (Amicon, Co. Cork, Ireland). To verify that labeling occurred in the extension Studies with Site-Specific Labeled HAMLET sequence of hLA-ext, a 200 fold dilution of the labeled hLA-ext was incubated with thrombin for 48 h. Cut and uncut protein samples were electrophoresed on 14% SDS-PAGE gel. A fluorescence signal was detected in a Hitachi FMBIOII Multi- View scanner with a 505-nm filter. To label hLA-ext and aD-hLA-ext with biotin, 2.3 mg of C2- keto-Gal glycosylated hLA-ext and aD -hLA-ext proteins were labeled in a 300-ml volume containing 166 mM sodium acetate, pH 4.9, and 3 mM aminooxy-biotin (Dojindo Laboratories, Japan). The reaction mixture was incubated overnight at room temperature. Excess biotin was removed by washing with 20 mM Tris-HCl (pH 8.0) in Amicon Ultra centrifugal filters with 10,000 MWCO for hLA-ext, and 3,000 MWCO for aD-hLA-ext (Millipore, MA). To detect the labeled protein, 10-, 50-, and 80-ng samples were separated on an SDS-PAGE. Protein was transferred to a nitrocellulose membrane (Invitrogen), blocked with 5% nonfat dry milk, 0.2% Tween 20 for 1 h at room temperature, and incubated for 1 h with 1:4000 diluted streptavidin conjugated with horseradish peroxidase (GE Healthcare, Piscataway, NJ) in a PBS solution containing 3% BSA and 0.02% Tween 20. After four 5- min washes with blocking solution, the membrane was incubated with HRP substrate ECL Western Blotting Analysis System (GE Healthcare) and exposed to Kodak BioMax light film. To detect the labeling in the extension tag, samples were treated with thrombin. Control samples remained untreated. Labeled hLA-ext was converted to molten globule and complexed with OA as described below. Preparation of hLA–OA, unlabeled and labeled hLA-ext– OA complexes a-LA–OA complexes were prepared by the method described by Kamijima et al. [30], with following modification: briefly, a 210-mM solution of recombinant hLA was incubated overnight with 5 mM EGTA and dialyzed against 5 mM EGTA/20 mM Tris-HCl (pH 8.0) at room temperature to remove calcium. EGTA was removed by a second dialysis overnight at room temperature against 20 mM Tris-HCl (pH 8.0). OA (Fluka, Buchs, Switzerland) was initially dissolved in 20% ethanol/80% 20 mM Tris-HCl (pH 8.0) solution and further diluted in 20 mM Tris-HCl (pH 8.0) buffer to give a 24-mM stock solution of OA. A solution of the molten globule protein (210 mM) in 20 mM Tris-HCl (pH 8.0) was mixed with OA stock solution (in a 1:10 molar ratio of protein to OA), heated for 10 min at 60oC, and cooled to room temperature. Excess OA was removed by mild centrifugation. Further, protein (complexed or molten globule alone) was precipitated with NaCl (final concentration 1M) and adjusted to a final pH of 3.5 with 1 M HCl. After 4 h on ice or overnight at 4oC, the precipitated complex was re-dissolved in 20 mM Tris-HCl (pH 8.0) and dialyzed overnight against 20 mM Tris-HCl (pH 8.0) to remove excess NaCl. For His-hLA samples, after removal of calcium, complexation with OA was performed as described above. Determination of protein to OA ratio Protein concentration of the complex was determined by the Bradford method, and OA concentration was determined by HPLC- ESI-MS/MS technique using a HPLC (Agilent 1100 series) coupled to triple quadrupole mass spectrometer (TSQ Quantum Discovery Max from Thermo Scientific) utilizing the single-ion monitoring technique in negative ionization mode [41]. For quantitation of OA, stable isotopic OA containing 18 [13C]-labeled carbon atoms was used as the internal standard, and was purchased from Cambridge Isotope Laboratories (Andover, MA). OA was monitored at m/z 281.2, and PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e26093