1The Protein Engineering Network of Centres of Excellence, 750 Heritage Medical Research Centre, Edmonton, AB, T6G 2S2 and 2Department of Microbiology and Immunology and 3The Biotechnology Laboratory, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada 4A.B.Boraston and B.W.McLean contributed equally to this work 5Present address: Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8P 5C2, Canada 6Present address: Twinstrand Therapeutics, 8081 Lougheed Highway, Burnaby, BC, V5A 1W9, Canada
7 To whom correspondence should be addressed. E-mail: rajw{at}interchange.ubc.ca
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Abstract |
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Keywords: carbohydrate-binding module/fusion proteins/N-glycosidase
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Introduction |
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The novelty of this work lies in the properties of the fusion proteins and in the use of cellulose for their immobilization. The fusion proteins behave as if bound irreversibly to the cellulose beads. The beaded cellulose is a very stable and easily manipulated matrix for simultaneous purification and immobilization of the fusion proteins.
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Materials and methods |
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The sources of materials were as follows: kanamycin, RNAseB, RNaseA, MT500 Perloza cellulose beads, Schiff's reagent and gentisic acid, Sigma; EndoF1, Novagen/ Calbiochem; PNGaseF and Pwo DNA polymerase, Boehringer Mannheim; HiTrap Chelating HP and HisTrap HP, Amersham Biosciences; restriction endonucleases, New England BioLabs; isopropyl-ß-D-thiogalactoside (IPTG), Rose Scientific; factor X, Hematologic Technologies; and agar, Difco.
Bacterial strains and plasmids
The bacterial strains used were C.meningosepticum American Type Culture Collection (ATCC) 33958 and E.coli strains R1360 for cloning and BL21 DE3 and Tuner DE3 for protein production. The plasmids used were pTugK (Graham et al., 1995) and pTugK-H6-IEGR-CBM2a (McLean et al., 2000
) for fusion construction and pET-28a(+) (Novagen) for protein production.
Growth of bacteria
Cultures of E.coli were grown in TYP medium (16 g of yeast extract, 16 g of tryptone, 5 g of NaCl, distilled water to 1 l; 10 ml of sterile 750 mM K2HPO4 added after autoclaving) containing 100 mg ml1 kanamycin. Solid medium contained 1.5% agar.
Construction of gene fusions
DNA sequences encoding mature EndoF1 and PNGaseF were amplified by polymerase chain rection (PCR) (Perkin-Elmer GeneAmp) with a suspension of the lyophilized cells received from ATCC as template. Each tube contained 20 or 100 pmol of each primer (Table I), 2 µl of cell suspension, 0.4 mM of each deoxynucleoside triphosphate, 2.5 units of Pwo DNA polymerase and 10 ml of the 10x buffer supplied with enzyme, in a total volume of 100 µl. The conditions were 25 cycles of 95°C for 30 s, 63°C for 45 s, with the temperature for this step being reduced by 0.5°C for each successive cycle and 72°C for 90 s; then 10 cycles of 95°C for 30 s, 55°C for 45 s and 72°C for 90 s. The primers were designed to add an AatII site to the 5' end of each coding sequence and a NotI site at the 3' end (Table I). An 400 bp DNA fragment encoding CBM2a followed by a prolinethreonine linker (PT linker) and a factor Xa (FXa) cleavage site was amplified from 50 ng of pTUGK-H6-IEGR-CBM2a, using the same PCR protocol: the primer for the 5' end of the coding sequence was complementary to the vector upstream of the multiple cloning site; the primer for the 3' end of the coding sequence added a short linker peptide and an AatII site to the sequence (Table I). The EndoF1-encoding fragment (50 pg) was fused to the CBM2A-encoding fragment (50 pg) by extension with Pwo DNA polymerase without added primers, using five cycles of 95°C for 30 s and 72°C for 3 min, after which 20 pmol of each flanking primer were added and the gene fusions amplified by 30 cycles of 95°C for 45 s, 55°C for 45 s and 72°C for 90 s. The product was purified by agarose gel electrophoresis, digested with NheI and NotI, then ligated into pTugK which had been digested with the same enzymes, yielding pTugKCBMEndoF1. The PNGase encoding fragment was digested with AatII and NotI and ligated to the larger of the two fragments produced by digestion of pTugKCBMEndoF1 with the same enzymes, yielding pTugKCBMPNGaseF. E.coli R1360 was transformed with the final products. Transformants were selected on kanamycin and screened by restriction enzyme digestion of their plasmids. The DNA sequences of the constructs were confirmed using a primer complementary to the 3' end of the sequence encoding CBM Xyn10A. The gene fusions were sub-cloned into pET-28a(+), then transformed into E.coli BL21 DE3 for production of the fusion proteins.
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Cultures (2x250 ml) of E.coli Tuner DE3 carrying pET28a(+)CBMEndoF1 or pET28a(+)CBMPNGaseF were grown with shaking at 30°C for 46 h to OD600 nm 1.0. IPTG was added to 0.1 mM and incubation continued for a further
18 h at 25°C. The cells were collected by centrifugation (4°C, 5000 rpm, 20 min), resuspended in 50 ml of binding buffer (0.5 M NaCl, 20 mM TrisHCl pH 8.0) containing 5 mM imidazole and ruptured by passage twice through a French pressure cell. Cell debris was removed by centrifugation (4°C, 15000 rpm, 30 min). The supernatant was made 37.5 mM in imidazole, then passed through a column of HiTrap Chelating HP or HisTrap HP (5 ml bed volume). The column was washed with a linear gradient of imidazole (37.550 mM, total volume 75 ml) in binding buffer. The fusion proteins were eluted with a linear gradient of imidazole (50250 mM, total volume 125 ml) in loading buffer. Fractions (5 ml) were screened for the fusion protein by SDSPAGE. CBMPNGaseF was eluted with
150 mM and CBMEndoF with
200 mM imidazole. Fractions containing the fusion protein were pooled and the buffer was changed by ultrafiltration to 100 mM potassium phosphate, pH 6.0 for CBMEndoF1 and pH 8.0 for CBMPNGaseF. Protein concentrations were estimated by measuring OD280nm.
Denaturation of RNaseB
RNaseB (100 µg) in 50 µl of denaturation buffer (100 mM potassium phosphate pH 8.0, 0.1% SDS, 50 mM ß-ME) was boiled for 5 min. After cooling to room temperature, Nonidet P40 or Triton X-100 was added to a final concentration of 0.75%.
Deglycosylation in solution
Reaction mixtures (50 µl total volume) contained 10 pmol of N-glycosidase, 100 µg of substrate (substrate to enzyme ratio 200, w/w) and 100 mM potassium phosphate (pH 6.0 for CBMEndoF1, pH 8.0 for CBMPNGaseF). The mixtures were incubated at 37°C. Samples of 7.5 µl were removed at intervals, added to 7.5 µl of loading buffer and boiled for 3 min, then 5 µl (equivalent to 5 µg of substrate) were analyzed by SDSPAGE.
Immobilization of the fusion proteins
CBMEndoF1 or CBMPNGaseF (100 pmol, 5 µg) was adsorbed on 0.5 mg of Perloza beads in 500 µl 100 mM potassium phosphate (pH 6.0 for CBMEndoF1, pH 8.0 for CBMPNGaseF), 0.02% sodium azide for 1 h at 4°C. The beads were washed with 1.0 ml of the appropriate buffer then resuspended in 50 µl of the same buffer.
Deglycosylation by the immobilized fusion proteins
Reaction mixtures were as for deglycosylation in solution, but using 5 µl of the appropriate Perloza bead suspension (equivalent to 10 pmol of fusion protein). The mixtures were incubated in a tube roller at 37°C. Samples were removed at intervals and processed as described above.
Stability of the immobilized fusion proteins
Fusion protein (5 µg) was adsorbed on 1 mg of Perloza beads in 500 µl of 100 mM potassium phosphate (pH 6.0 for CBMEndoF1, pH 8.0 for CBMPNGaseF), 0.02% sodium azide for 30 min at 4°C. The beads were washed (3x1.0 ml) with the same buffer, then resuspended in 200 µl of either the same buffer or denaturation buffer with 0.75% NP-40 or Triton X-100. The suspensions were incubated overnight at 4 or 37°C. The beads were washed once with 1.0 ml of the same buffer, resuspended in 200 µl of 100 mM potassium phosphate, pH 6.0 or 8.0, and 10 µl of the final suspensions were screened by SDSPAGE.
Glycan analysis
The glycans released from RNaseB by CBMEndoF1, both in solution and immobilized on cellulose, were analyzed by matrix-assisted, laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The reaction mixture was set up as described above, but using 25 mM ammonium acetate pH 5.0 instead of phosphate buffer. After incubation, protein in the reaction mixture was precipitated by the addition of 60 µl of cold 95% ethanol, then removed by centrifugation (14 000 r.p.m., 4°C, 10 min). The supernatant was lyophilized and the residue dissolved in 5 µl of acetonitriletrifluoroacetic acid (7:3, v/v). Samples of the solution were diluted 1:1, 1:2, 1:5 and 1:10 with the same solvent. A 2 µl sample of each dilution was mixed with 2 µl of gentisic acid (10 mg/ml in the same solvent). A 1 µl sample of each mixture was spotted on the target for spectroscopy. Spectra were obtained with a Ciphergen SELDI-Massphoresis system.
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Results |
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The fusion proteins encoded by pTugKCBMEndoF1 and pTugKCBMPNGaseF were designed to be transported to the periplasm in E.coli. The leader peptides of Xyn10A and endoglucanase 6A (Cel6A) from C.fimi facilitate the transport of some fusion proteins to the periplasm of E.coli, with high-level production sometimes leading to leakage of the fusion protein into the culture medium (Greenwood et al., 1994). Unfortunately, the levels of the CBMN-glycosidase fusion proteins obtained were only 10100 µg/l of culture supernatant. Therefore, the gene fusions were sub-cloned into pET28a(+) in order to obtain intracellular production of the proteins. This had the added advantage of replacing the FXa site following the hexahistidine sequence with a thrombin site, allowing removal of the hexahistidine tag without removal of the CBM (Figure 1). The amino acid sequence of CBMEndoF1 deduced from the nucleotide sequence of the gene fusion differed from the published sequence of EndoF1 at two positions: a serine for an asparagine at position 63 and an alanine for a valine at position 218. Neither of these residues is involved in forming the active site of the enzyme (Van Roey et al., 1994
). The fusion protein was as active as native EndoF1 on RNaseB and active also on a-amylase (Rydberg et al., 1999), so the mutations were not corrected. There were also two non-conservative changes in the amino acid sequence of PNGase F in the CBMPNGase F fusion. These were corrected by PCR-based site-directed mutation.
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The fusion proteins were purified by immobilized metal affinity chromatography (IMAC) on Ni2+-agarose (Figure 2). Use of the hexahistidine tag for purification is preferable to use of the CBM because desorption of CBMfusion proteins from cellulose may require denaturing conditions. The yields of purified protein were 40 and 20 mg/l of culture for CBMPNGaseF and CBMEndoF1, respectively. The yield of CBMPNGaseF was comparable to that reported for production of PNGaseF by E.coli (Lemp et al., 1990).
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RNaseB, which carries a single N-linked glycan of the high-mannose type (Fu et al., 1994), was used as the substrate for evaluating the fusion proteins. Removal of the glycan is detected by SDSPAGE, the glycan-free form of the protein, RNaseA, migrating faster than the native protein.
The activities of the fusion proteins were compared with those of equal molar equivalents of the native enzymes, taking into account the higher molecular weights of the fusion proteins. CBMEndoF1 in solution had comparable activity to EndoF1 on native RNaseB (Figure 3). Neither PNGaseF nor CBMPNGaseF in solution had detectable active on native RNaseB under the assay conditions used; they had comparable activity on denatured RNaseB (Figure 3). Denaturation of RNaseB increases its susceptibility to PNGaseF 844-fold (Chu, 1986).
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Like the native enzyme, CBMPNGaseF is active on denatured but not native protein substrates. Therefore, the stability was determined of the binding of CBMPNGaseF to Perloza beads under conditions used to denature proteins prior to deglycosylation. It remained bound to Perloza beads after incubation at 37°C overnight in standard and denaturing buffers (Figure 6). A potential advantage of CBM fusion proteins is their stability when bound to Perloza beads (Kwan et al., 2002). The fusion proteins bound to Perloza beads lost little or no activity when stored for 28 days in phosphate buffer at 4°C (Figure 7).
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The oligosaccharides released from RNaseB by CBMEndoF1 in solution and immobilized on cellulose were identical and matched the profile obtained for deglycosylation by the native enzyme (Fu et al., 1994 ). This confirmed that immobilization did not impede access to the substrate and showed that the oligosaccharides did not come from the cellulose (Figure 8). The analysis was not extended to PNGaseF because the reagents used to denature RNaseB interfered with the subsequent MALDI-TOFMS of the released glycans.
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Discussion |
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The tags have an insignificant effect on glycosidase activity in solution. There is a slight reduction in activity on immobilization, but this is to be expected. Immobilization by chemical coupling with reagents such as CNBr can result in a >60% reduction in the specific activity of an enzyme, largely as a consequence of restricted mass transfer (Wiseman, 1985). Immobilization on cellulose via a CBM has little effect on the efficiency of mass transfer. Furthermore, it appears to stabilize the fusion partner (Kwan et al., 2002
) and as seen with CBMEndoF1. If only the denatured form of a glycoprotein is sensitive to CBMPNGase, the glycosidase can be used in solution and then removed or the denaturants removed or diluted before use of the immobilized enzyme.
CBMEndoF1 and CBMPNGase are potentially very useful tools for glycoprotein analysis. CBMEndoF1 was used to deglycosylate human pancreatic -amylase, produced in Pichia pastoris prior to crystallization (Rydberg et al., 1999). Furthermore, their stability and production in good yield by E.coli make them relatively inexpensive. Compared with the glutathione-S-transferase N-glycosidase fusions (Grueninger-Leitch et al., 1996
), they have the advantage that cellulose is cheap and it can be used without modification as a matrix for immobilization.
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Acknowledgements |
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Received June 21, 2005; revised July 21, 2005; accepted August 3, 2005.
Edited by Pang-Chui Shaw
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