N-Glycosidase–carbohydrate-binding module fusion proteins as immobilized enzymes for protein deglycosylation

Emily M. Kwan1,2, Alisdair B. Boraston1,2,3,4,5, Bradley W. McLean1,2,3,4,6, Douglas G. Kilburn1,2,3 and R. Antony J. Warren1,2,7

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A carbohydrate-binding module (CBM) was fused to the N-termini of mannosyl-glycoprotein endo-ß-N-acetylglucosaminidase (EndoF1) and peptide N-glycosidase F (PNGaseF), two glycosidases from Chryseobacterium meningosepticum that are used to remove N-linked glycans from glycoproteins. The fusion proteins CBM–EndoF1 and CBM–PNGaseF also carry a hexahistidine tag for purification by immobilized metal affinity chromatography after production by Escherichia coli. CBM–EndoF1 is as effective as native EndoF1 at deglycosylating RNaseB; the glycans released by both enzymes are identical. Like native PNGaseF, CBM–PNGaseF is active on denatured but not on native RNaseB. Both fusion proteins are as active on RNaseB when immobilized on cellulose as they are in solution. They retain activity in the immobilized state for at least 1 month at 4°C. The hexahistidine tag can be removed with thrombin, leaving the CBM as the only affinity tag. The CBM can be removed with factor Xa if required.

Keywords: carbohydrate-binding module/fusion proteins/N-glycosidase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The glycan and protein moieties of glycoproteins must be separated prior to their characterization. Two endo-glycosidases from Chryseobacterium (formerly Flavobacterium) meningosepticum are used routinely to cleave the N-linked glycans from a protein: mannosyl-glycoprotein endo-ß-N-acetylglucosaminidase (EndoF1) (Elder and Alexander, 1982Go) and peptide N-glycosidase F (PNGaseF) (Plummer et al., 1984Go). EndoF1 is a glycosidase, hydrolyzing the glycosidic bond between the N-acetylglucosamines (GlcNAcs) of the chitobiose core of high-mannose and biantennary hybrid-type but not complex oligosaccharides, leaving a single GlcNAc on the asparagine. PNGaseF is an amidase, hydrolyzing the bond between the chitobiose core and the asparagines of high-mannose, biantennary hybrid-type and complex oligosaccharides (Tarentino et al., 1985Go). Both enzymes may be more active on the denatured than on the native form of a glycoprotein, in which case producing enough of the deglycosylated protein for crystallography may require appreciable quantities of enzyme. Commercially available preparations of the enzymes are expensive. Active EndoF1 and PNgaseF were produced in Escherichia coli either fused to maltose-binding protein (Kuhn et al., 1995Go; Rao et al., 1999Go) or to glutathione-S-transferase (Grueninger-Leitch et al., 1996Go), facilitating both their purification from cell extracts and their easy removal from the target protein after deglycosylation. The fusions were not tested in the immobilized state. Immobilization of an enzyme may stabilize it and allow its repeated use, in effect reducing its cost. We reported previously the construction and properties of a carbohydrate-binding module-factor Xa fusion protein (CBM–FXa) that appears to bind irreversibly to cellulose, is both stable and active on protein substrates when bound and can be used at effective substrate:CBM–FXa ratios of >104 (Kwan et al., 2002Go). The properties of CBM–FXa, together with the cheapness and stability of cellulose, led us to construct similar derivatives of both EndoF1 and PNGaseF, again using CBM2a of xylanase 10A from Cellulomonas fimi, as described in this paper. This CBM is an effective fusion partner for both protein purification (Rodriguez et al., 2004Go) and enzyme immobilization (Ong et al., 1992; Kwan et al., 2002Go). Both enzymes were engineered because of their different specificities. EndoF is most active between pH 4.0 and 6.0 and PNGaseF between pH 7.0 and 9.0. Both are optimally active in 100 mM sodium or potassium phosphate buffers, but 25 mM ammonium acetate can be used for EndoF1, especially around pH 5.0. All of these conditions are compatible with the binding of CBM2a to cellulose.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Materials

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., 1995Go) and pTugK-H6-IEGR-CBM2a (McLean et al., 2000Go) 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 ml–1 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 proline–threonine 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 pTugK–CBM–EndoF1. The PNGase encoding fragment was digested with AatII and NotI and ligated to the larger of the two fragments produced by digestion of pTugK–CBM–EndoF1 with the same enzymes, yielding pTugK–CBM–PNGaseF. 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.


View this table:
[in this window]
[in a new window]
 
Table I. Primers used in the construction of the gene fusions

 
Production and purification of the fusion proteins

Cultures (2x250 ml) of E.coli Tuner DE3 carrying pET28a(+)–CBM–EndoF1 or pET28a(+)–CBM–PNGaseF were grown with shaking at 30°C for 4–6 h to OD600 nm {approx} 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 Tris–HCl 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 (37.5–50 mM, total volume 75 ml) in binding buffer. The fusion proteins were eluted with a linear gradient of imidazole (50–250 mM, total volume 125 ml) in loading buffer. Fractions (5 ml) were screened for the fusion protein by SDS–PAGE. CBM–PNGaseF was eluted with ~150 mM and CBM–EndoF 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 CBM–EndoF1 and pH 8.0 for CBM–PNGaseF. 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 CBM–EndoF1, pH 8.0 for CBM–PNGaseF). 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 SDS–PAGE.

Immobilization of the fusion proteins

CBM–EndoF1 or CBM–PNGaseF (100 pmol, ~5 µg) was adsorbed on 0.5 mg of Perloza beads in 500 µl 100 mM potassium phosphate (pH 6.0 for CBM–EndoF1, pH 8.0 for CBM–PNGaseF), 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 CBM–EndoF1, pH 8.0 for CBM–PNGaseF), 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 SDS–PAGE.

Glycan analysis

The glycans released from RNaseB by CBM–EndoF1, 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 acetonitrile–trifluoroacetic 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.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Construction of the gene fusions

The fusion proteins encoded by pTugK–CBM–EndoF1 and pTugK–CBM–PNGaseF 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., 1994Go). Unfortunately, the levels of the CBM–N-glycosidase fusion proteins obtained were only 10–100 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 CBM–EndoF1 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., 1994Go). 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 CBM–PNGase F fusion. These were corrected by PCR-based site-directed mutation.



View larger version (8K):
[in this window]
[in a new window]
 
Fig. 1. Generalized structure of the CBM–N-glycosidase fusion proteins. CBM2a, carbohydrate-binding module from xylanase 10A of Cellulomonas fimi; catalytic module, EndoF1 or PNGase sequence.

 
Purification of the fusion proteins

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 CBM–fusion proteins from cellulose may require denaturing conditions. The yields of purified protein were 40 and 20 mg/l of culture for CBM–PNGaseF and CBM–EndoF1, respectively. The yield of CBM–PNGaseF was comparable to that reported for production of PNGaseF by E.coli (Lemp et al., 1990Go).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2. Purity of the fusion proteins. The proteins were purified by IMAC as described in Materials and methods. Purity was assessed by SDS-PAGE. Lanes: 1, CBM–EndoF1 (2 µg); 2, CBM–PNGaseF (2 µg); 3, molecular weight standards (kDa).

 
Activity of the fusion proteins

RNaseB, which carries a single N-linked glycan of the high-mannose type (Fu et al., 1994Go), was used as the substrate for evaluating the fusion proteins. Removal of the glycan is detected by SDS–PAGE, 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. CBM–EndoF1 in solution had comparable activity to EndoF1 on native RNaseB (Figure 3). Neither PNGaseF nor CBM–PNGaseF 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, 1986Go).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3. Deglycosylation of RNaseB by N-glycosidases in solution. Reaction mixtures were as described in Materials and methods. Reaction mixtures corresponding to lanes 3–6 contained native RNaseB; those corresponding to lanes 7–10 contained denatured RNaseB. Samples were removed at intervals and deglycosylation assessed by SDS–PAGE. Lanes: 1, molecular weight standards (kDa); 2, RNaseB untreated; 3 and 4, EndoF1 0.5 and 1.0 h, respectively; 5 and 6, CBM–EndoF1 0.5 and 1.0 h, respectively; 7 and 8, PNGaseF 0.5 and 1.0 h, respectively; 9 and 10, CBM–PNGaseF 0.5 and 1.0 h, respectively; 11, RNaseA.

 
Perloza beaded cellulose is an excellent cellulose matrix for immobilizing CBM–fusion proteins (Kwan et al., 2002Go). Unlike many forms of cellulose, it does not compress when used in columns, maintaining a good flow rate. When bound to Perloza, both fusion proteins were as active as the corresponding soluble forms (Figure 4). CBM–EndoF1 was inactive on native FX (data not shown), possibly because of the structures of the glycans. For example, native EndoF1 does not remove glycans with a fucose linked {alpha}(1–6) to the first N-acetylglucosamine residue (van Roey et al., 1994Go). The soluble and immobilized forms of CBM–PNGaseF deglycosylated factor X (Figure 5). Schiff staining (not shown) indicated that the deglycosylation was incomplete. Any O-linked glycans would not be removed by the enzyme.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4. Deglycosylation of RNaseB by immobilized glycosidases. CBM–EndoF1 and CBM–PNGaseF were immobilized on Perloza beads and reaction mixtures were set up as described in Materials and methods. Reaction mixtures corresponding to lanes 3 and 4 contained native RNaseB, those corresponding to lanes 5 and 6 contained denatured RNaseB. Samples were removed at intervals and deglycosylation assessed by SDS-PAGE. Lanes: 1, molecular weight standards (kDa); 2, RNaseB; 3 and 4, CBM–EndoF1 0.5 and 1.0 h, respectively; 5 and 6, CBM–PNGaseF 0.5 and 1.0 h, respectively; 7, RNaseA.

 


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5. Deglycosylation of factor X by CBM–PNGaseF. Reaction mixtures were set up as for the deglycosylation of RNaseB but with FX as the substrate. Deglycosylation was assessed by SDS–PAGE. The gel was stained with Coomassie Brilliant Blue. Lanes: 1, molecular weight standards; 2, untreated FX; 3 Perloza-bound CBM–PNGaseF for 18 h; 4, CBM–PNGaseF in solution for 18 h.

 
Stability of immobilized CBM–PNGaseF

Like the native enzyme, CBM–PNGaseF is active on denatured but not native protein substrates. Therefore, the stability was determined of the binding of CBM–PNGaseF 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., 2002Go). 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).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6. Stability of CBM–PNGaseF immobilized on Perloza. CBM–PNGaseF was bound to Perloza. Samples of the beads were suspended in different buffers as described in Materials and methods, then incubated overnight at 4 or 37°C. The beads were washed, resuspended in 100 mM phosphate buffer pH 8.0 and the protein remaining bound to the beads assessed by SDS–PAGE. Lanes: 1, beads in phosphate buffer stored overnight at 4°C; 2, beads in phosphate buffer incubated overnight at 37°C; 3, beads in denaturation buffer with NP40 incubated overnight at 37°C; 4, beads in denaturation buffer with Triton X-100.

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7. Stability of immobilized fusion proteins during storage at 4°C. Samples of the same suspension of Perloza beads with bound CBM–EndoF1 and PNGaseF used for the experiment shown in Figure 4 were stored at 4°C for 1 month, then screened for activity in the same manner. Deglycosylation was assessed by SDS–PAGE. Lanes: 1, molecular weight standards; 2, RNaseB untreated; 3–6, treated with immobilized CBM–EndoF1 for 0.5, 1.0, 1.5 and 2.0 h, respectively; 7–10, denatured RNaseB treated with immobilized CBM–PNGaseF for 0.5, 1.0, 1.5 and 2.0 h, respectively; 11, RNaseA.

 
Glycan analysis

The oligosaccharides released from RNaseB by CBM–EndoF1 in solution and immobilized on cellulose were identical and matched the profile obtained for deglycosylation by the native enzyme (Fu et al., 1994Go ). 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.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 8. MALDI-TOF mass spectra of glycans released from RNaseB by CBM–EndoF1. RNaseB was treated with CBM–EndoF1 in solution (A) or immobilized on Perloza (B). The released glycans were recovered and analyzed by mass spectrometry as described in Materials and methods. M4–M9 are core N-linked glycans having 4–9 mannose residues, respectively, in addition to the N-acetylglucosamine remaining attached to the substrate.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
A hexahistidine tag facilitates purification of an enzyme by IMAC and its removal from substrate after use. Ni2+-agarose is unsuitable, however, as a matrix for immobilizing the enzyme. A CBM tag can be used to immobilize an enzyme and to facilitate its removal after use in solution. Under the appropriate conditions, however, it behaves as if irreversibly bound after adsorption to crystalline cellulose and desorption may require strongly denaturing conditions, depending on the nature of its fusion partner (Gilkes et al., 1992Go; McLean et al., 2000Go). Although this may limit its usefulness as an affinity tag for purification, the ‘irreversible’ binding makes it a very useful tag for immobilizing enzymes (Ong et al., 1991Go; Kwan et al., 2002Go; this work). The CBM–EndoF1 and CBM–PNGase fusion proteins described here carry both a hexahistidine tag for purification and removal from substrate and a CBM for removal from substrate or immobilization on cellulose. We used the hexahistidine tag to prepare the soluble fusion proteins for characterization. For practical purposes, when the enzymes are to be used bound to cellulose purification and immobilization can be carried out in a single step using the CBM tag.

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, 1985Go). 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., 2002Go) and as seen with CBM–EndoF1. If only the denatured form of a glycoprotein is sensitive to CBM–PNGase, the glycosidase can be used in solution and then removed or the denaturants removed or diluted before use of the immobilized enzyme.

CBM–EndoF1 and CBM–PNGase are potentially very useful tools for glycoprotein analysis. CBM–EndoF1 was used to deglycosylate human pancreatic {alpha}-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., 1996Go), they have the advantage that cellulose is cheap and it can be used without modification as a matrix for immobilization.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank the Protein Engineering Network of Centres of Excellence and CBD Technologies, Inc., for financial support.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Chu,F.K. (1986) J. Biol. Chem. 261, 172–177.[Abstract/Free Full Text]

Elder,J.H. and Alexander,S. (1982) Proc. Natl Acad. Sci. USA, 79, 4540–4544.[Abstract/Free Full Text]

Fu,D., Chen,L. and O'Neill,R.A. (1994) Carbohydr. Res., 261, 173–186.[CrossRef][ISI][Medline]

Gilkes,N.R., Jervis,E., Henrissat,B., Tekant,B., Miller,R.C.,Jr and Kilburn,D.G. (1992) J. Biol. Chem. 267, 6743–6749.[Abstract/Free Full Text]

Graham,R.W., Greenwood,J.M., Warren,R.A.J., Kilburn,D.G. and Trimbur,D.E. (1995) Gene, 158, 51–54.[CrossRef][ISI][Medline]

Greenwood,J.M., Gilkes,N.R., Miller,R.C.,Jr, Kilburn,D.G. and Warren,R.A.J. (1994) Biotech. Bioeng., 44, 1295–1305[CrossRef][ISI]

Grueninger-Leitch,F., D'arcy,A., D'arcy,B. and Chène,C. (1996) Protein Sci., 5, 2617–2622.[Abstract/Free Full Text]

Kuhn,P., Guan,C., Cui,T., Tarentino,A.L., Plummer,T.H.,Jr and Van Roey,P. (1995) J. Biol. Chem., 270, 29493–29497.[Abstract/Free Full Text]

Kwan,E.M., Guarna,M.M., Boraston,A.B., Gilkes,N.R., Haynes,C.A., Kilburn,D.G. and Warren,R.A.J. (2002) Biotechnol. Bioeng., 79, 724–732.[CrossRef][ISI][Medline]

Lemp,D., Haselbeck,A. and Klebl,F. (1990) J. Biol. Chem., 265, 15606–15610.[Abstract/Free Full Text]

McLean,B.W., Bray,M.R., Boraston,A.B., Gilkes,N.R., Haynes,C.A. and Kilburn,D.G. (2000) Protein Eng., 13, 801–809.[CrossRef][ISI][Medline]

Ong,E., Gilkes,N.R., Miller,R.C.,Jr, Warren,R.A.J. and Kilburn,D.G. (1991) Enzyme Microb. Technol., 13, 59–65.[CrossRef][ISI][Medline]

Plummer,T.H.,Jr, Elder,J.H., Alexander,S., Phelan,A.W. and Tarentino,A.L. (1984) J. Biol. Chem., 259, 10700–10704.[Abstract/Free Full Text]

Rao,V., Cui,T., Guan,C. and Van Roey,P. (1999) Protein Sci., 8, 2338–2346.[Abstract/Free Full Text]

Rodriguez,B., Kavoosi,M., Koska,J., Creagh,A.L., Kilburn,D.G. and Haynes,C.A. (2004) Biotechnol. Prog. 20, 1479–1489.[CrossRef][ISI][Medline]

Rydberg,E.H. et al. Protein Sci., 8, 635–643.

Tarentino.,A.L., Gomez,C.M. and Plummer,T.H.,Jr (1985) Biochemistry, 24, 4665–4671.[CrossRef][ISI][Medline]

Van Roey,P., Rao,V., Plummer,T.H.,Jr and Tarentino,A.H. (1994) Biochemistry, 33, 13989–13996.[CrossRef][ISI][Medline]

Wiseman,A. (ed.) (1985) Handbook of Enzyme Biotechnology. 2nd edn. Ellis Horwood, Chichester.

Received June 21, 2005; revised July 21, 2005; accepted August 3, 2005.

Edited by Pang-Chui Shaw





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
18/10/497    most recent
gzi055v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Request Permissions
Google Scholar
Articles by Kwan, E. M.
Articles by Warren, R. A. J.
PubMed
PubMed Citation
Articles by Kwan, E. M.
Articles by Warren, R. A. J.