Glycobiology Advance Access originally published online on December 17, 2007
Glycobiology 2008 18(2):137-144; doi:10.1093/glycob/cwm131
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Communication |
Fishing for lectins from diverse sequence libraries by yeast surface display – An exploratory study
3 Department for Molecular Biomedical Research, Unit for Fundamental and Applied Molecular Biology, VIB
4 Department of Molecular Biology, Ghent University
5 Department for Molecular Biomedical Research, Unit for Molecular Glycobiology, VIB
6 Department of Biochemistry, Physiology and Microbiology, Ghent University, B-9052 Ghent, Belgium
1 To whom correspondence should be addressed: Tel: +32-9-331-3617; Fax: +32-9-331-3502; e-mail: Nico.Callewaert{at}DMBR.UGent.be
Received on May 9, 2007; revised on November 28, 2007; accepted on November 30, 2007
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Abstract |
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The establishment of a robust technology platform for the expression cloning of carbohydrate-binding proteins remains a key challenge in glycomics. Here we explore the utility of using yeast surface display (YSD) technology in the interaction-based lectin cloning from complete cDNA libraries. This should pave the way for more detailed studies of protein–carbohydrate interactions. To evaluate the performance of this system, lectins representing three different subfamilies (galectins, siglecs, and C-type lectins) were successfully displayed on the surface of Saccharomyces cerevisiae and Pichia pastoris as a-agglutinin and/or
-agglutinin fusions. The predicted carbohydrate-binding activity could be detected for three out of five lectins tested (galectin-1, galectin-3, and siaoadhesin). For galectin-4 and E-selectin, no specific carbohydrate-binding activity could be detected. We also demonstrate that proteins with carbohydrate affinity can be specifically isolated from complex metazoan cDNA libraries through multiple rounds of FACS sorting, employing multivalent, fluorescent-labeled polyacrylamide-based glycoconjugates. Key words: expression cloning / lectin / Pichia pastoris / Saccharomyces cerevisiae / yeast surface display
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Introduction |
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TopAbstractIntroductionResults and discussionMaterials and methodsConflict of interest statementReferences |
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Lectins are carbohydrate-binding proteins involved in numerous biological processes (Gabius et al. 2002
). Moreover, they are essential tools in the emerging field of glycomics, allowing specific detection of target analytes, hence fulfilling functions analogous to that of antibodies in proteomics.
Phage display represents a major advance in the antibody-engineering field, but is of limited use in the interaction cloning of proteins with carbohydrate-binding activity (Yamamoto et al. 1999; Ravn et al. 2004). First, because protein-carbohydrate interactions depend largely on multivalency (Ravn et al. 2004), the monovalent display format, which is applied in most cases, may not be optimal for dealing with carbohydrate antigens. Second, a prokaryotic expression host has an unpredictable but frequently strong expression bias against many eukaryotic proteins. To complement the lectin cloning tools offered by phage display with a eukaryotic counterpart, we rely on yeast surface display (YSD) (Schreuder et al. 1993; Boder and Wittrup 1997). This technology was originally developed to enhance secretion efficiency, stability, and affinity of proteins (Boder et al. 2000; Holler et al. 2000; Shusta et al. 2000), but has not been used to identify proteins with carbohydrate affinity. The multivalent YSD format intrinsically mimics the natural multivalent presentation of lectins on cells (Figure 1A), and could largely overcome the technical problems observed with other (prokaryotic) protein display platforms that have been used for lectin cloning (Ravn et al. 2004). We build on the successes of the YSD format that were obtained in the antibody-engineering field. Here it proved successful in both the isolation of scFv's from large nonimmune libraries (Feldhaus et al. 2003) and the maturation of these scFv's toward higher affinities. Currently, the yeast display method has yielded the highest affinity (48 fM) for any antibody (Boder et al. 2000). We foresee similar application for YSD in the cloning and engineering of lectins.
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Results and discussion |
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TopAbstractIntroductionResults and discussionMaterials and methodsConflict of interest statementReferences |
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To evaluate the possibility of using YSD to study protein-carbohydrate interactions, we displayed on the surface of Saccharomyces cerevisiae three galectins from different subfamilies and possessing different natural oligomerization properties (galectin-1: dimeric, galectin-3 and -4: monomeric) (system outline: Figure 1A). Galectins are soluble β-galactoside-binding lectins (Barondes et al. 1994
) that are expressed as surface receptors when introduced in the YSD system. Likewise, when expressing cDNA libraries, all soluble receptors become cell surface localized. Hereby, the AGA1-AGA2 complex links these proteins to the cell wall. Using fluorescence-activated cell sorting (FACS) analysis with a fluorescein isothiocyanate (FITC)-labeled anti-V5-tag mAb, we determined that a large fraction of cells displayed high levels of the full-size proteins (mean fluorescence intensity is up to 100-fold higher than that of nonexpressing cells) (Figure 2A–C). Subsequently, by FACS analysis we demonstrated the interaction between the surface-displayed galectins and multivalent, LacNAc-containing, fluorescent-labeled polyacrylamide-based glycoconjugates (LacNAc-PAA-FITC) (Galanina et al. 1998). We demonstrated that galectin-1 and -3 interact specifically with LacNAc-PAA-FITC (Figure 2F and G). However, only a few cells displaying galectin-4 showed binding to this conjugate (Figure 2H), which concurs with the notion that galectin-4 has a weak affinity for LacNAc (instead it binds specifically to 3'-O-sulfated Galβ1,3-GalNAc) (Ideo et al. 2002). However, the lower intrinsic surface expressibility, compared to galectin-1 and -3, could also contribute to this. As can be deduced from Figure 1B, the minimum expression threshold that allows detection of carbohydrate binding is not reached for galectin-4 (Figure 2).
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To broaden the scope of our technology, we displayed the carbohydrate recognition domain (CRD) of sialoadhesin, which belongs to the siglec family of animal lectins. Despite high levels of display (Figure 2D), no lectin activity could be detected on the yeast surface (Figure 2I). Therefore, we switched to Pichia pastoris, which is an outstanding host for the production of heterologous proteins and often gives higher yields than S. cerevisiae. Moreover, the availability of glycoengineered Pichia strains that produce hybrid and complex N-glycans (Vervecken et al. 2004
; Wildt and Gerngross 2005) should allow the detection of mannooligosaccharide-binding lectins, which might otherwise become quenched and undetectable in this yeast-based approach due to the presence of large amounts of cell wall mannan. These lectins are instead expected to agglutinate the yeast cells, which produce high-mannose glycans. Strikingly, in a Pichia strain with abolished hypermannosylation (Vervecken et al. 2004), surface display levels of both galectin-1 and sialoadhesin a-agglutinin fusions were lower than that in S. cerevisiae (compare Figure 2K and L with Figure 2A and D) and no sugar-PAA-FITC binding could be detected (Figure 2N and O). In contrast, in Pichia, surface display of sialoadhesin -agglutinin fusion resulted in increased display levels and improved 3'SiaLac-PAA-FITC binding as compared to S. cerevisiae (compare Figure 2M and P with Figure 2E and J). This might reflect the need for correct topological orientation of the CRD to obtain full carbohydrate binding. Table I gives an overview of the system performance in both yeasts for the different lectins that were tested.
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Several reasons could account for the absence of binding. First, we suggest that the absence of carbohydrate binding in the low-expressing strains is due to the lack of avidity. Indeed, by double staining cells for full-size protein expression and carbohydrate binding, we showed that the number of cells that bind to sugar-PAA-FITC increased exponentially for higher display levels (Figure 1B). This is convincing evidence for the existence of a protein-specific expression level threshold that allows an avidity-stabilized "velcro-type" interaction. Alternatively, the lack of detection for some of the tested lectins could be due to their inability to move sufficiently freely on the cell surface (since they are linked to the immobile β-glucan of the outer cell wall of the yeast cells), which prevents them from the crucial clustering needed for multivalency and increased avidity. In this respect, combining the YSD format with carbohydrate microarrays based on neoglycolipid (NGL) technology (Feizi and Chai 2004
) holds great promise, because these arrays confer a mobility to the lipid-tailed glycans very similar to that in natural membranes.
Having both C- and N-terminal fusion protein display systems at hand in both S. cerevisiae and (glyco-engineered) P. pastoris increases the versatility of the developed technology and thus the chances for success. Nevertheless, difficult cases remain, as we observed with E-selectin. When the CRD of E-selectin was displayed as -/a-agglutinin fusions, no binding with SiaLeX- PAA-FITC could be detected in either yeasts despite high V5-epitope display levels (data not shown). This indicates that, apart from the requirement for avidity, other factors, such as protein folding and surface stability, might render surface lectin activity undetectable in this yeast-based approach.
With these potentially versatile lectin surface display systems at our disposal, we wished to test the feasibility of an interaction-based cloning of lectin genes. To verify that cells displaying a functional lectin can be sorted from a large heterogeneous population using FACS, we performed a model selection by sorting cells with surface display of galectin-3 from a background of control cells. The experiment was carried out with cell ratios of 1:10, 1:100 and 1:1000, and the composition of the postenrichment cell mixture was determined by polymerase chain reaction (PCR) screening. The results showed that strong enrichment of up to 200-fold can be obtained in one round of flow cytometric sorting when cells displaying galectin-3 were mixed with a large excess (1000-fold) of cells expressing the control plasmid (see Table II).
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Having established this efficient system for FACS-based sorting, we proceeded to provide proof of principle by cloning lectins from several complex metazoan cDNA libraries (for experimental setup see Figure 1C). We constructed and independently surface displayed a cDNA library derived from HepG2 human hepatoma cells, a chicken liver cDNA library, and a human fetal brain cDNA library, each containing >106 independent clones. We focused on isolating proteins that bind lactose or N-acetyl-lactosamine (LacNAc). For the isolation of transmembrane lectins we wish to rely on random primed libraries, since this will allow isolation of lectin domains that still exhibit carbohydrate binding activity. To increase the specificity of sorting, cells were double stained for fusion protein display (using the in-frame cloned Xpress-tag) and carbohydrate binding (Figure 1C). An overview of the system performance for the expressed cDNA libraries is given in Table III. About 0.01 to 0.02% of the cells were sorted out in the first round. Flow cytometric analysis after two rounds of sorting revealed that 5–10% of the cells bound to the LacNAc-PAA-FITC conjugate (as exemplified for the HepG2 cDNA library in Figure 1C), which approaches the frequency of LacNAc-PAA-FITC-stainable cells in a pure galectin-3-displaying population. To fully eliminate false positives, individual clones were assessed for nonspecific binding to a non-glycan-substituted conjugate that was otherwise structurally identical. Several of the sorted clones exhibited affinity towards PAA-FITC and were thus eliminated (Figure 3D–F). Sequences of the true positive clones revealed the presence of three full-length and three truncated cDNA variants of galectin-3. One of the latter, containing the C-terminal part of galectin-3 starting at Leu-120, still specifically bound LacNAc-PAA-FITC (Figure 3A–C). This confirms earlier reports on the minimal lactose-binding domain of galectin-3 (Moriki et al. 1999
). Also, three different clones expressing full-length galectin-1 were isolated. Sorting of the chicken liver cDNA library led to isolation of a clone expressing CG-16, which encodes a 16-kDa β-galactoside-binding protein (Sakakura et al. 1990). It is important to note that this strategy of double sorting followed by a test at the clonal level for nonspecific binding to the noncarbohydrate-substituted probe did not introduce any false positives into DNA sequencing. This testifies to the efficiency of our methodology, and our overall results indicate that this interaction-based cloning method can very efficiently identify lectin genes from complex cDNA libraries.
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To summarize, we describe a novel interaction-based methodology for cloning lectins from complex cDNA libraries. We have shown that, given functional expressibility of a lectin in S. cerevisiae or P. pastoris, yeasts displaying this lectin can be efficiently isolated from large libraries without false positives. We now envisage exploiting this efficient lectin display technology, in combination with the recently developed glycan arrays (Feizi and Chai 2004
), to develop lectin sequence variants with narrower specificity and higher affinity. The existence of specific disease-related glycosylation changes (Ragupathi 1996; Callewaert et al. 2004) drives this search for the development of lectins as effective diagnostic and therapeutic tools (Ravn et al. 2004). |
Materials and methods |
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TopAbstractIntroductionResults and discussionMaterials and methodsConflict of interest statementReferences |
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Used strains
S. cerevisiae – EBY100 (Invitrogen, Carlsbad, CA)
P. pastoris – GlycoswitchMan5 (Vervecken et al. 2004)
Vector construction and transformation
S. cerevisiae. The human galectin-3 cDNA was amplified by PCR from a HepG2 cDNA library using oligonucleotides 5'-GGATCCATGGCAGACAATTTTTCGCTC-3' and 5'-GGG- CCCTATCATGGTATATGAAGCACTGG-3'. The amplified fragment was cloned BamHI/ApaI into pYD1 (Invitrogen). Galectin-1 and galectin-4 coding sequences were obtained from the mammalian gene collection (http://mgc.nci.nih.gov/). The human galectin-1 CDS was PCR-amplified using oligonucle- otides 5'-GGGGGTACCGATGGCTTGTGGTCTGGTCGC3' and 5'-GGGCTCGAGGTCAAAGGCCACACATTTG-3', and galectin-4 using 5'-GGGGAATTCATGGCCTATGTCCCCGC- ACC-3' and 5'-GGGCTCGAGGATCTGGACATAGGACAA- GGTG-3'. Amplified DNA of galectin-1 and galectin-4 was cloned KpnI/XhoI and EcoRI/XhoI, respectively, into pYD1. The obtained plasmids were transformed into the S. cerevisiae strain EBY100 (Boder and Wittrup 1997) (Invitrogen) as described (Gietz et al. 1995). This strain has a genomically integrated copy of a galactose-inducible expression cassette for S. cerevisiae AGA1.
A fragment encoding the V-set domain of human sialoadhesin (corresponding to the N-terminal 119 amino acids) was PCR-amplified from a genomic MGC clone using oligonucleotides 5'-ATCAGTGGATCCTCATGGGGCGTCT- CCAGTCC-3' and 5'-CAGTCTCGAGCTCCTCTGTTACTG- TGACCAAGG-3'. A fragment encoding the lectin-EGF domain of human E-selectin (corresponding to the N-terminal 157 amino acids of the mature protein) was amplified from a HepG2 cDNA library using oligonucleotides 5'-CAGTGGAT- CCTGGTCTTACAACAACTCCAC-3' and 5'-CAGTCTCGA- GCACAATTTGCTCACACTTGAG-3'. DNA amplified from sialoadhesin and E-selectin was cloned BamHI/XhoI into pYD1. The obtained plasmids, pYD1sialoadhesin and pYD1E-selectin, were transformed to S. cerevisiae strain EBY100 as described (Gietz et al. 1995).
A fragment encoding the C-terminal 320 amino acids of the yeast SAG1 was amplified by PCR from genomic DNA of S. cerevisiae strain InvScI (Invitrogen) using oligonucleotides 5'-CCGGAATTCAGCGCCAAAAGCTCTTTTATC-3' and 5'- CCCAAGCTTTTAGAATAGCAGGTACGAC-3'. A fragment encoding V5-tagged sialoadhesin was PCR-amplified using pYD1sialoadhesin as a template and oligonucleotides 5'-GGA- TATCTCATGGGGCGTCTCCAGTCC-3' and 5'-CCGGAA- TTCCGTAGAATCGAGACCG-3'. The amplified DNA was cloned EcoRV/EcoRI into NaeI/EcoRI-opened pPIC92 (BCCM/LMBP Plasmid Collection, Ghent University, Belgium; accession no. 3369) to give pPIC92sialoadhesin. Next, prepro-sialoadhesin-V5 was cut BamHI/EcoRI from pPIC92- sialoadhesin and cloned together with the EcoRI/HindIII-digested SAG1 fragment into the BamHI/HindIII-opened pYX133 (Ingenius Co. Oxford, UK) to give pYSDsia- loadhesin. pYSDE-selectin was constructed similarly starting from a PCR fragment encoding V5-tagged E-selectin using pYD1E-selectin as a template and oligonucleotides 5'-GGATATCTGGTCTTACAACACCTCCACG-3' and 5'-CCGGAATTCCGTAGAATCGAGACCG-3'. The obtained plasmids were transformed in S. cerevisiae strain InvScI as described (Gietz et al. 1995).
P. pastoris. Galectin-1, sialoadhesin and E-selectin AGA2 fusions were placed under control of the AOX1 promotor and transformed to Pichia GS115 strain with an integrated copy of both pGlycoswitchM5 and pPICZAGA1 (containing S. cerevisiae AGA1 under control of the AOX1 promotor) (Pieter Jacobs, in preparation).
S. cerevisiae SAG1 was PCR amplified from pYSDE-selectin using oligonucleotides 5'-CCGGAATTCAGCGCCA- AAAGCTCTTTTATC-3' and 5'-ATAGTTTAGCGGCCGCT- TAGAATAGCAGGTACGAC-3' and cloned EcoRI/NotI into pPIC92sialoadhesin and pPIC92E-selectin to give pPSD- sialoadhesin and pPSDE-selectin. Obtained plasmids were transformed in Pichia strain GS115 with an integrated copy of pGlycoswitchM5 (Vervecken et al. 2004) as described (Cregg and Russel 1998).
Surface expression and FACS analysis
S. cerevisiae. Yeast cells were grown overnight to saturation in the synthetic D-glucose complete medium (SDC) composed of 2% dextrose/0.67% yeast nitrogen base/1% casamino acids. After dilution, the cells were grown for further 6 h to an OD600 of 0.5. To induce protein expression, cells were pelleted by centrifugation, washed twice with distilled water, resuspended to an OD600 of 0.5 in 2% galactose/0.67% yeast nitrogen base/1% casamino acids, and incubated at 20°C for 40 h.
P. pastoris. Yeast cells were grown for 24 h in the buffered glycerol-complex medium (BMGY). To induce protein expression, cells were pelleted by centrifugation, washed twice with distilled water, resuspended in the buffered methanol-complex medium (BMMY) and grown for another 24 h.
Surface expression of the full-length proteins was demonstrated using an antibody against the V5-epitope, which was intentionally fused C-terminally to the coding sequences during cloning. After induction, 107 cells in 1 mL phoshphate-buffered saline (PBS) (pH 7.2), supplemented with 0.5 mg/mL bovine serum albumin (BSA), were incubated with 1 µL/mL anti-V5 antibody (1 µg/µL; Invitrogen), washed with PBS/BSA, and incubated with 1 µL/mL Alexa fluor 488-labeled goat antimouse IgG (1 µg/µL; Molecular Probes). After washing twice with PBS/BSA, the cells were analyzed by flow cytometry.
Carbohydrate binding assay
To test the carbohydrate binding ability of the obtained strains, 107-induced yeast cells were incubated with 100 pmol of sugar-PAA-FITC (GlycoTech) for 30 min at 4°C in PBS/BSA. After washing twice with PBS/BSA, the cells were resuspended in PBS/BSA and analyzed by flow cytometry.
Single step enrichment of cells displaying galectin-3 using LacNAc-PAA-FITC and flow cytometric sorting
Two strains, one containing the empty pYD1 vector (and thus expressing the Aga2p-Aga1p heterodimer without the Aga2p fusion partner) and one containing pYD1galectin-3 were grown and induced separately. After induction, the cells were washed twice in PBS/BSA, and cell numbers determined by OD600 measurement. Cells expressing the Aga2p-galectin-3 fusion protein were subsequently mixed with empty vector control cells at 1:10, 1:100, and 1:1000 ratios. For each ratio, 
107 cells were incubated with 100 pmol Lac-PAA-FITC sorted using a FACS Calibur fluorescence-activated cell sorter (Becton Dickinson, Sunnyvale, CA) for above-background fluorescence, and collected on a 0.22-µm filter. Sorted cells were recuperated by washing the filter and plated on the selective medium (SDC-Trp). To determine the enrichment efficiency of fluorescent cell sorting, DNA was prepared from 100 randomly chosen colonies (Elder et al. 1983). Galectin-3-specific primers were used to check for the presence of a galectin-3 cDNA insert.
cDNA cloning and construction of a SfiI/NotI-adapted yeast surface display vector
The templates for cDNA synthesis were commercially available poly A+ RNA from human fetal brain and chicken liver (Clontech). Poly A+ RNA extracted from a pool of human HepG2 cells was also used for library construction. The construction of cDNA libraries was carried out essentially as described (Chen et al. 2003). A pYD1 vector (Invitrogen) was adapted for the directional cloning of SfiI/NotI-terminated cDNA as follows. Using the oligonucleotides 5'-CCGGAATTCGGCCAAAAA- GGCCTCGAGG-3' and 5'-GGGGATCCTCTAGGCGGCCG- CTAGGCCTC-3', a PCR fragment was amplified from pSCGAL10-SN (BCCM/LMBP Plasmid Collection, Ghent University, Belgium; accession no. 2471), and after EcoRI/NotI digestion, it was ligated into the EcoRI/NotI-opened pYD1 vector. In this way, the SfiI restriction site downstream of the AGA2 coding sequence in pYD1 is followed by a stuffer fragment and a newly introduced NotI restriction site.
As it was observed that reverse transcription at times terminates at very specific sites, two more vectors were derived from pYD1SfiI/NotI, to ensure fusion of the cDNA to AGA2 in three reading frames. One and two base pairs, respectively, were inserted in pYD1SfiI/NotI+1 and pYD1SfiI/NotI+2, downstream of the AGA2 coding sequence and upstream of the SfiI site. For this, we used the Quickchange® Site Directed Mutagenesis Kit (Stratagene). The SfiI/NotI-digested cDNA was ligated to an equimolar mixture of SfiI/NotI-digested pYD1SfiI/NotI, pYD1SfiI/NotI+1, and pYD1SfiI/NotI+2, and the ligation mix was transformed into electrocompetent E. coli MC1061 cells. Transformed cells were grown on LB plates supplemented with ampicillin (100 µg/mL) and grown overnight at 37°C. The colonies were collected by washing off the plates, and plasmid DNA was prepared and transformed to the S. cerevisiae strain EBY100 (Gietz et al. 1995). Generally, libraries of more than 106 yeast clones were obtained.
cDNA library screening using LacNAc-PAA-FITC
About 107 yeast cells from the surface display library in 1 mL of PBS were incubated with anti-Xpress antibody (1 µg/mL) followed by Alexa fluor 647 R-phycoerythrin goat antimouse secondary antibody (Molecular Probes), allowing the detection of cells expressing a fusion protein in the FL3 channel. Then the yeast cells were washed, resuspended in PBS (pH 7.2) supplemented with 0.5 mg/mL BSA, and incubated with 100 pmol LacNAc-PAA-FITC for 20 min at 4°C. The cells were washed twice and diluted with PBS supplemented with 0.5 mg/mL BSA immediately before sorting. The cells that exhibited the highest fluorescence were isolated as described above. When needed, two subsequent rounds of selection were performed; after sorting, the cells were collected by washing the filter and plated. After that cells were recultured and induced as before. The cells that were collected in the final FACS round were plated to isolate individual clones, which were then analyzed further.
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Conflict of interest statement |
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TopAbstractIntroductionResults and discussionMaterials and methodsConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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The authors thank A. Bredan for carefully editing the manuscript. S.R. is supported by a Ph.D. grant from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). The Ghent University Research Council and the Fund for Scientific Research-Flanders (FWO) are acknowledged for grants to R.C.; N.C. is a Marie Curie Excellence Grant recipient (EU, 6th Framework Program).
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Footnotes |
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2 Both first authors contributed equally.
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Abbreviations |
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BMGY, buffered glycerol-complex medium; BMMY, buffered methanol-complex medium; BSA, bovine serum albumin; CRD, carbohydrate recognition domain; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; LacNAc, N-acetyl-lactosamine; PAA, polyacrylamide; PBS, phosphate-buffered saline, PCR, polymerase chain reaction; SDC, synthetic D-glucose complete medium; SiaLAc, sialyllactose; YSD, yeast surface display
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References |
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