High-level expression of recombinant Neisseria CMP-sialic acid synthetase in Escherichia coli Marie-France Karwaski, Warren W.Wakarchuk, and Michel Gilbert* Institute for Biological Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ont., Canada K1A 0R6 Received 14 November 2001, and in revised form 19 January 2002 Abstract The CMP-sialic acid synthetase (CMP-Neu5Ac, synthetase) is responsible for the synthesis of CMP-Neu5Ac, which is the donor used by sialyltransferases to attach sialic acid to acceptor hydroxyl groups in various polysaccharides, glycolipids, and glycopro- teins.Since CMP-Neu5Ac is unstable and relatively expensive, the CMP-Neu5Ac synthetase is valuable for the preparative enzy- matic synthesis of sialylated oligosaccharides.We made a construct to over-express the Neisseria meningitidis CMP-Neu5Ac synthetase in Escherichia coli.The recombinant enzyme was expressed at very high level (over 70,000 U/L) in a soluble form.It was purified by a sequence of anion-exchange chromatography and gel filtration with an overall yield of 23% (specific activity 220U/mg). The purified CMP-Neu5Ac synthetase was used in the gram-scale synthesis of CMP-Neu5Ac. �� 2002 Elsevier Science (USA).All rights reserved. N-Acetylneuraminic acid (Neu5Ac or sialic acid) is an important molecule in biological interactions.In mam- malian systems, sialylated oligosaccharides have been shown to be involved in cell���cell recognition, cell dif- ferentiation, and various receptor���ligand interactions [1].Sialylated glycoconjugates are also found in bacte- rial pathogens, where they are structures that mimic oligosaccharides found in mammalian glycolipids and are probably used to evade the host immune response [2].Sialyltransferases transfer sialic acid from the acti- vated sugar nucleotide CMP-Neu5Ac to acceptor oli- gosaccharides found on glycoproteins, glycolipids or polysaccharides.The CMP-Neu5Ac synthetase (EC 2.7.7.43) is the enzyme that catalyzes the formation of the CMP-Neu5Ac by the following reaction: CTP �� Neu5Ac ! CMP--Neu5Ac �� PPI Since CMP-Neu5Ac is unstable and relatively ex- pensive, the CMP-Neu5Ac synthetase is valuable for the preparative enzymatic synthesis of sialylated oligosac- charides.It can also be used to charge sialic acid analogs to synthesize the corresponding sialo-oligosaccharide analogs.CMP-Neu5Ac synthetases have been cloned from various bacterial and mammalian sources [3���11]. Previously we reported the cloning and expression of the Neisseria meningitidis CMP-Neu5Ac synthetase in Escherichia coli at a production level of 360 U/L [3].This construct included a poly-His tail at the C-terminus and it was purified by metal a���nity chromatography.We also made a fusion between the Neisseria CMP-Neu5Ac synthetase and a-2,3-sialyltransferase which yielded 11,500 U/L of CMP-Neu5Ac synthetase activity and was used to synthesize various sialylated oligosaccharides [12].Knorst and Fessner [11] recently published a re- combinant E. coli construct that produced 7500 U/L of the Neisseria CMP-Neu5Ac synthetase.In this work we present a construct optimized for the expression of the Neisseria CMP-Neu5Ac synthetase that produces over 70,000 U/L and a simple procedure for the purification of the native enzyme. Materials and methods Plasmid construction The N. meningitidis 406Y CMP-Neu5Ac synthetase gene (GenBank #U60146) was amplified using SYNTM-F1 as the 50 primer (50-CTTAGGAGGTCAT Protein Expression and Purification 25 (2002) 237���240 www.academicpress.com * Corresponding author.Fax: 613-941-1327. E-mail address: michel.gilbert@nrc.ca (M. Gilbert). 1046-5928/02/$ - see front matter �� 2002 Elsevier Science (USA).All rights reserved. PII: S1046-5928(02)00004-9

ATGGAAAAACAAAATATTGCGGTTATAC-30, the NdeI site is shown in italics), SYNTM-R1 as the 30 primer (50-TCGAAGATCTTAGCTTTCCTTGTGATT AAGAATG-30, the BglII site is shown in italics), 100 ng of N. meningitidis 406Y chrDNA as template and Vent DNA polymerase (New England Biolabs, Mississauga, Canada).The PCR product was digested with NdeI and BglII and cloned in pT7-7 cut with NdeI and BamHI to obtain the plasmid pNSY-01.The construct used in this work was obtained by transferring the CMP-Neu5Ac synthetase from pT7-7 to a modified version of pCW- ori+ [13] using the NdeI and SalI sites to obtain the plasmid pNSY-05 (Fig.1). Expression of the CMP-Neu5Ac synthetase E. coli AD202 (E. coli Genetic Stock Center, CGSC #7297) was transformed with pNSY-05.The enzyme production was tested in 1-L cultures.We inoculated 1L of 2X YT medium with 10 mL of an overnight culture of E. coli AD202 (pNSY-05).The culture was grown at 37 ��C until A600 �� 0:3, induced with 1mM IPTG, and grown for an additional 6h.The cells from 1mL sam- ples were sonicated and analyzed for CMP-Neu5Ac synthetase activity.The production of CMP-Neu5Ac synthetase activity was measured in three separate cul- tures grown on different days. Purification of the CMP-Neu5Ac synthetase Cells from 10mL cultures were centrifuged and re- suspended in 1 mL of 20 mM Tris, pH 8.3. The cells were sonicated 4 1min at power 2 (50% cycle) using a Sonifier 450 (Branson Ultrasonics, Danbury, CT).The cell debris were removed by centrifugation at 14,000 rpm in a microfuge.The supernatant was loaded onto a 1-mL MonoQ column (Amersham Pharmacia Biotech, Baie d���Urfe e, Canada) equilibrated with 20mM Tris, pH 8.3. Bound proteins were eluted with a gradient of 0���0.7 M NaCl in starting buffer at a flow rate of 0.5 mL/ min for a total of 15mL.The fractions eluting between 0.22 and 0.28 M NaCl were pooled, concentrated by ultrafiltration (10,000 molecular weight cut-off mem- brane), and applied to a 10mm 30cm Superose 12 column (Pharmacia Amersham Biotech).The Superose 12 column was developed with 20mM Tris, pH 8.3, at a flow rate of 0.5 mL/min. Enzyme assay The CMP-Neu5Ac synthetase activity was assayed using CTP, Neu5Ac, Gal-b1,4-GlcNAc-FCHASE (6- (5-fluorescein-carboxamido)-hexanoic acid succimidyl ester) and a purified fusion of the N. meningitidis a-2,3- sialyltransferase (MalE-NST, unpublished) in a coupled assay that measured the production of Neu5Aca-2,3- Gal-b1,4-GlcNAc-FCHASE.One unit of activity was defined as the amount of enzyme that produces 1 lmol of product in 1min.The reaction mix included 0.5 mM Gal-b-1,4-GlcNAc-FCHASE, 3mM CTP, 3mM Neu5Ac, 4mU of a-2,3-sialyltransferase, 100 mM Tris, pH 7.5, 10mM MgCl2, and 0.2 mM DTT. The reactions were incubated at 37 ��C for 5 min, stopped by the ad- dition of acetonitrile (25% final concentration), and di- luted with H2O to get 10���15lM final concentration of the FCHASE-labelled compounds.The samples were analyzed by capillary electrophoresis performed using the separation and detection conditions as described previously [14].The peaks from the electropherograms were analyzed using manual peak integration with the P/ACE Station software. CMP-Neu5Ac synthesis We dissolved 1g (1.8mmol) CTP and 0.62 g (2 mmol) Neu5Ac in 11.5 mL of 65mM Hepes and the pH was adjusted to 8 with NaOH.We added MgCl2 and DTT to final concentrations of 50 and 0.2 mM, respectively. The reaction was started by the addition of 100 U of CMP- Neu5Ac synthetase and the total incubation time was 20h at 37 ��C.We added additional CMP-Neu5Ac syn- thetase (63 U each time) after 5 and 16h.Before adding fresh enzyme, we centrifuged the white precipitate (Mg- pyrophosphate), adjusted the pH to 8, and added Fig.1.Structure of the plasmid (pNSY-05) used to express the CMP- Neu5Ac synthetase from Neisseria meningitidis.The CMP-Neu5Ac synthetase gene was cloned in a modified version of pCWori+ [13], a plasmid that has three sequential IPTG-inducible promoter upstream of the insert. 238 M.-F. Karwaski et al. / Protein Expression and Purification 25 (2002) 237���240