Recombinant DNA techniques were carried out using standard methods and commercially available materials. PCR amplifications were performed using either Taq Ready-to-Go beads (GE Healthcare) or a proofreading DNA polymerase, Phusion HF, purchased from New England BioLabs. Transformants were screened by either restriction digestion of plasmid minipreps or directly by PCR. Freshly picked colonies for PCR screening were boiled in diethyl pyrocarbonate water for 5 min and centrifuged. The supernatant was mixed with 1 μl of appropriate primers and Ready-to-Go PCR beads and then amplified in a thermocycler and analyzed on agarose gels.
Construction of expression plasmids.
Plasmids and primers used for our constructions are described in Table .
The pWV234 NusA-PST plasmid encodes an amino-terminal NusA fusion with the NmC PST (synE). The synE gene was amplified from chromosomal DNA isolated from N. meningitidis group C strain P2181 with the forward primer CPST LIC FOR and the reverse primer CPST LIC REV and Taq Ready-to-Go PCR beads. The amplified fragment was purified and ligated into the expression vector pET44 Ek/LIC containing the nusA gene (Novagen) as described in the manufacturer's instructions.
For WV235 SUMO-PST, the synE gene was amplified as described above by using plasmid pWV234 as template with forward primer CPST-SUMO Met FOR and reverse primer CPST-SUMO REV. The amplified fragment was ligated in the pETSUMO TOPO plasmid as recommended by Invitrogen.
For pWV237, N. meningitidis group B synD was amplified from chromosomal DNA isolated from strain H355 using primers NmB rev HindIII and NmB directional for. The resulting fragment was ligated into the pET151 TOPO vector from Invitrogen.
For pWV241, the Gateway system of Invitrogen was used to construct a chimera of the maltose binding protein (MBP) gene, malE, and the NmC PST gene, synE. The synE gene was introduced into an entry vector as follows. The synE gene was amplified from pWV234 with the forward primer NmC PST FWD pENTR TOPO, the reverse primer NmC PST BamHI REV, and the thermostable polymerase reagent Phusion HF master mix from Finnzymes. The purified fragment was ligated into the Invitrogen pENTR/TEV/D-TOPO plasmid as recommended by the manufacturer.
pWN602 malE Gateway destination vector.
The plasmid pN-WWW was a gift of Warrren Wakarchuk, National Research Council, Canada, and is based on pCWOri+ from the same source. pN-WWW was digested with NdeI and HindIII and converted to blunt ends with mung bean nuclease and calf intestine alkaline phosphatase. The Gateway cassette B was inserted into the blunt end site as described by Invitrogen. The resulting plasmid, pWN602, contained the Gateway B cassette at the 3′ end of the malE gene.
For pWV243 MBP-PST, the syn E (NmC PST) gene in pWV241 was used as the insert gene for the LR clonase (Invitrogen) reaction. pWN602 was used as the destination vector for pWV243. The protein expressed by this plasmid has an MBP at the amino-terminal end of NmC PST separated by a tobacco etch virus (TEV) protease cleavage site.
For pWV246, the HaloTag fusion plasmid was constructed by inserting the synE gene into the expression plasmid pFN22K HaloTag Flexivector (Promega). The synE gene was amplified from the plasmid pWV243 with the Phusion Hi-Fidelity enzyme mix (New England BioLabs) using the primers NmC PST Flexivector forward and NmC PST Flexivector reverse.
For pWV239, the NmB PST gene, synD, was cloned into the malE-containing Gateway vector pWN602 in a fashion similar to that described for pWV243 above. The synD gene was first amplified using primers NmB directional for and NmB rev HindIII and then inserted in pENTR/TEV-TOPO. The resulting plasmid, designated pWV238, was combined with pWN602 in an in vitro recombination reaction mixture to yield pWV239. The resulting plasmid expresses a MalE-PST chimera with MalE and the amino terminus of NmCPST separated by a TEV protease cleavage site.
For pWV248, the NmC PST synE gene was amplified with pWV234 as PCR template and NmCPST fwd pENTR TOPO and MengC PST rev as primers. The resulting NmC PST synE was ligated into pET151TOPO as described by the manufacturer.
The domain swap mutants of NmB and NmC PST were constructed by two methods. The plasmid pWV245 was constructed with the QuikChange II XL kit (Stratagene). The sequence encoding the amino terminus of SynD (nucleotides 1 to 439) was amplified using the primers DSnmB FOR and DSnmB REV with the NmB PST plasmid pWV239 as a template. The resulting fragment was purified and used as a mutagenic primer with the QuikChange II XL kit to mutate the NmC PST expression plasmid pWV243. This procedure requires significant areas of homology in the regions to be swapped. Because of this requirement for homology, the QuikChange method was not suitable for swapping the desired shorter region of synD
. A modification of the method of Stemmer and Morris (25
) was used to construct the other synD
chimera, although inverse PCR was not performed (4
). Briefly, nucleotides 1 to 322 were amplified with a primer that included nucleotides 322 of synD
in a Bsa1 cleavage site and a pET151 vector-based primer, including its Bsa1 site from pWV237. Similarly, nucleotides 322 to 1479 of synE
were amplified with a primer that included nucleotides 322 of synE
in a Bsa1 cleavage site and a synE
primer that included its Bsa1 site from pWV248. The fragments were isolated, cut with Bsa1, and ligated together. The resulting hybrid fragment was ligated into the Bsa1 sites of pWV248 to yield plasmid pWV249. Plasmid pWV249 was sequenced and found to have a deletion error in the region of one of the primers used in its construction. The error was corrected using a QuikChange Lightning mutagenesis kit to give pWV250. The sequences of all plasmids were confirmed by sequencing in the CBER Core Facility.
The plasmids pWV245 and pWV250 were transformed into E. coli BL21(DE3) cells, and the polysialyltransferase activities of cell lysates were determined as described below.
The protein concentration was determined using the Bio-Rad dye binding reagent. Purified enzyme fractions were analyzed by SDS-PAGE (Invitrogen) and stained with Coomassie blue. HaloTag proteins were labeled as described above, with a tetramethylrhodamine (TAMR)-HaloTag, and analyzed by SDS-PAGE with fluorescence detection.
Polysialyltransferase expression and purification.
The NusA polysialyltransferase chimera was expressed by transformation of plasmid pWV234 into BL21(DE3) Star and preparation of an overnight inoculum in 25 ml of LB containing 200 μg/ml ampicillin. The culture in LB broth (1.5 liters) containing 50 μg/ml ampicillin was inoculated and shaken at 30°C until the absorbance at 600 nm reached 1.2. The culture was induced by addition of 360 mg isopropyl-β-d
-thiogalactopyranoside (IPTG) and shaking at 30°C for an additional 2 h 45 min. The cells were harvested by centrifugation at 10,000 × g
for 15 min, at 4 to 10°C, and stored at −80°C. Frozen cells were thawed and suspended in 10 ml 50 mM Tris, 10 mM MgCl2
in which was dissolved one EDTA-free protease inhibitor tablet (pH 7.5; Roche). The cell suspension was lysed in a French pressure cell and centrifuged for 12 min at 10,000 × g
, at 4 to 10°C. Membranes were separated from the cytosolic fraction by ultracentrifugation for 1 h at 114,000 × g
at 4°C. The membrane fraction was resuspended in 1.5 ml of cryo buffer (31
). The supernatant fraction was adjusted to 10% glycerol and stored at −80°C.
Polysialyltransferase constructs with a His tag epitope were purified from the supernatant by tumbling overnight at 2 to 8°C with 4 ml of Ni-nitrilotriacetic acid-agarose resin (Qiagen) in 50 mM Tris, 10 mM imidazole, pH 7.5. The suspension was poured into a column and washed at 0.5 ml/min with 50 mM Tris, 10 mM imidazole, 200 mM NaCl, pH 7.5. The protein was eluted with the same buffer containing 60 mM imidazole and then 250 mM imidazole, collecting 3-ml fractions. Fractions were assayed for activity using the paper chromatography assay described below. Peak activity fractions were combined and concentrated using a Centriplus YM10 apparatus from 4 ml to 1 ml. The concentrated enzymes was adjusted to 10% glycerol and stored at −80°C.
The SUMO-polysialyltransferase chimeric enzyme was purified from cells harboring the plasmid pWV235 by a similar procedure, except the culture was grown in LB with kanamycin.
The MalE-NmC polysialyltransferase was purified as follows. Plasmid pWV243 was transformed into AD202 cells, and the fresh transformants from the entire plate were suspended in a few milliliters of medium and used to inoculate 1.5 liters of rich broth (consisting of 15 g Bacto tryptone, 7.5 g yeast extract, 7.5 g NaCl) plus 3 g glucose and 100 μg/ml ampicillin (pH 7.2). Cells were shaken at 30°C for 4 h and then induced for 3 h with 400 mg IPTG. A cytosolic fraction was prepared as described above, and the enzyme content was purified on a 12-ml amylose resin (New England BioLabs) column. The amylose column was washed with 50 mM Tris, 200 mM NaCl, 1 mM EDTA, pH 7.4, and the protein was eluted with 10 mM maltose in the same buffer.
Preparation of fluorescent-tagged polysialyltransferase.
HaloTag-NmC PST was prepared by transforming pWV246 in BL21(DE3) STAR cells. The fresh transformants were used to inoculate 2 × 1.5 liters of LB-kanamycin and grown to an A600 of 0.7, then induced with 1 mM IPTG for 3 h. The cells were harvested and frozen in 25 ml 50 mM Tris, 25 mM MgCl2 (pH 8) overnight. The cell suspension was lysed in a French pressure cell, and cellular debris was removed by centrifugation at 10,000 × g for 15 min.
The lysate (25 ml) was labeled with 250 μl of TAMR-HaloTag substrate (10 to 20 μg/ml) as described by Promega. The membranes were separated from the soluble fraction by ultracentrifugation at 114,000 × g. The membrane pellet was resuspended in 4 ml cryoprotective buffer.
Gel filtration of HaloTag-NmC PST.
The soluble fraction described above was adjusted to 25% saturated ammonium sulfate and stirred at 4°C for 1 h. The pellet was collected by centrifuging at 15,000 rpm for 30 min and dissolved in 3 ml of 50 mM Tris, 25 mM MgCl2
(pH 8.0). Most of the enzyme activity in the soluble fraction was precipitated at 25% (NH4
saturation. The enzyme fraction was analyzed on a Superose 12 fast-protein liquid chromatography (FPLC) column (GE) on a Dionex Summit high-performance liquid chromatograph (HPLC) equipped with a Hitachi model L-7485 fluorescence detector. TAMR-labeled fractions were detected at an excitation wavelength of 555 nm and emission at 585 nm. Peak fractions were concentrated on Nanosep microconcentrators and assayed for polysialyltransferase activity as described previously (17
Assay for polysialyltransferase.
Polysialyltransferase activity was measured using a paper chromatography assay described previously (17
The linkage of the product of the chimera plasmids pWV245 and pWV250 was determined as follows. In a typical assay, the membrane fraction was incubated in a solution containing 22 mM Tris, 11 mM MgCl2, pH 8, 10 μg polysaccharide acceptor (NmC polysaccharide, colominic acid, or K92 polysaccharide) in 110 μl and 3 μl 100-μCi/ml 14C-labeled CMP-sialic acid (American Radiolabeled Chemicals). After a 1-h incubation, α-2,8-endoneuraminidase was added, and incubation at 37°C continued for 3 h, prior to analysis by paper chromatography. Control reaction mixtures were incubated for 3 h without endoneuraminidase.
NmC PST competition assay with unlabeled acceptors.
Lactosyl-boron dipyrromethene label (BODIPY) was sialylated with the bifunctional sialyltransferase CST II in a large-scale reaction volume (500 μl), and oligosialylated products were separated by anion exchange HPLC as described before (16
). Product peaks were collected and desalted using Sep Pak chromatography (Waters). The extent of sialylation was confirmed by matrix-assisted laser desorption ionization-mass spectrometery (data not shown). Trisialylated lactosyl-BODIPY (2.5 μM) was incubated with NmC PST (30 μg/ml) in the presence of CMP-NeuNAc (5 mM) and MnCl (40 mM) in 200 mM sodium cacodylate buffer (pH 8.0) at 37°C for 40 min. For competition experiments, the reaction was run in the presence of sialic acid trimer, hexamer, or GD3 (Calbiochem) at a concentration of 5 μM. All reactions were quenched by the addition of ethanol to 25%. Products were dried by using a Speedvac, brought up to 50 μl with H2
O, and subjected to HPLC analysis as previously described (18
Preparation of outer membrane vesicles.
Outer membrane vesicles (OMV) were prepared from cultures of mutant N. meningitidis
group B strains according to the procedure described by Frasch and Peppler (8
). Cultures were grown overnight on tryptic soy broth supplemented with yeast extract as described by Arakere et al. (2
). N. meningitidis
strain M7 lacking the ability to synthesize sialic acid and a synD
mutant lacking an active polysialyltransferase were obtained from David Stephens, Emory University (14
Detection of polysialic acid formation by ELISA.
NmC PST (13 μl) was incubated with 10 μl OMV and CMP-NeuNAc (33, 330, 1,000, or 2,000 μM) in a total of 30 μl as described previously (17
) for 1 h at 37°C. The assay mixture was then diluted to 400 μl with enzyme-linked immunosorbent assay (ELISA) coating buffer (2
), which was then used to coat Immulon 1B plates (100 μl/well) at room temperature overnight. The plates were washed and then incubated with mouse monoclonal IgG antibody Mcl2075 (against OAc+
) or Mcl2016 (against OAc−
) group C polysaccharide at 1:400 dilution (kindly donated by Marjorie Shapiro, FDA) (11
). Plates were developed with goat anti-mouse IgG-alkaline phosphatase conjugate (Sigma) diluted 1:2,000.