Biased mutagenesis in the N-terminal region by degenerate oligonucleotide gene shuffling enhances secretory expression of barley {alpha}-amylase 2 in yeast

Kenji Fukuda1,2,3, Malene H. Jensen4, Richard Haser4, Nushin Aghajari4 and Birte Svensson1,2,5

1Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, 2Biochemistry and Nutrition Group, BioCentrum-DTU, Technical University of Denmark, Søltofts Plads, Bldn. 224, DK-2800 Kgs. Lyngby, Denmark and 4Laboratoire de BioCristallographie, Institut de Biologie et Chimie des Protéines, UMR 5086–CNRS/UCBL, IFR 128 ‘BioSciences Lyon-Gerland’, 7 Passage du Vercors, F-69367 Lyon cedex 07, France

5 To whom correspondence should be addressed, at the Technical University of Denmark. E-mail: bis{at}biocentrum.dtu.dk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Recombinant barley {alpha}-amylase 1 (rAMY1) and 2 (rAMY2), despite 80% sequence identity, are produced in very different amounts of 1.1 and <0.05 mg/l, respectively, by Saccharomyces cerevisiae strain S150-2B. The low yield of AMY2 practically excludes mutational analysis of structure–function relationships and protein engineering. Since different secretion levels of AMY1/AMY2 chimeras were previously ascribed to the N-terminal sequence, AMY1 residues were combinatorially introduced at the 10 non-conserved positions in His14–Gln49 of AMY2 using degenerate oligonucleotide gene shuffling (DOGS) coupled with homologous recombination in S.cerevisiae strain INVSc1. Activity screening of a partial library of 843 clones selected six having a large halo size on starch plates. Three mutants, F21M/Q44H, A42P/A47S and A42P rAMY2, also gave higher activity than wild-type in liquid culture. Only A42P showed wild-type stability and enzymatic properties. The replacement is located to a ß->{alpha} loop 2 that interacts with domain B (ß->{alpha} loop 3) protruding from the catalytic (ß/{alpha})8-barrel. Most remarkably Pichia pastoris strain GS115 secreted 60 mg/l A42P compared with 3 mg/l of wild-type rAMY2. The crystal structure of A42P rAMY2 was solved and found to differ marginally from the AMY2 structure, suggesting that the high A42P yield stems from stabilization of the mature and/or intermediate form owing to the introduced proline residue. Moreover, the G to C substitution for the A42P mutation might have a positive impact on protein translation.

Keywords: codon usage/isozyme sequence-guided mutagenesis/stability/x-ray crystallography/yeast


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Barley (Hordeum vulgare) {alpha}-amylase 1 (AMY1) and 2 (AMY2) are de novo synthesized in seeds during germination and readily distinguished by their pI values. Although AMY1 and AMY2 share 80% sequence identity, they differ in enzymatic properties, effect of Ca2+ on activity, stability at elevated temperature and acidic pH and sensitivity to barley {alpha}-amylase/subtilisin inhibitor (BASI) (Søgaard and Svensson, 1990Go; Søgaard et al., 1991Go, 1993Go; Ajandouz et al., 1992Go; Rodenburg et al., 1994Go; Juge et al., 1995Go; Jensen et al., 2003Go). The structure of AMY2 was solved in the native state (Kadziola et al., 1994Go) and in complexes with acarbose, a pseudotetrasaccharide inhibitor (Kadziola et al., 1998Go) and with BASI (Vallée et al., 1998Go). AMY1 structures were determined recently in the native state and in complexes with thiomaltotetraose (Robert et al., 2003Go) and acarbose (Robert et al., 2005Go) and inactive mutant structures were determined in complexes with acarbose and maltoheptaose (Robert et al., 2005Go). Since the backbones of AMY1 and AMY2 superimpose perfectly, the differing behaviors of the isozymes probably stem from local differences in structure and surface electrostatic potential (Robert et al., 2002Go; Bak-Jensen et al., 2004Go). Germinated seeds contain 10–20 times more AMY2 than AMY1, whereas recombinant AMY1 (rAMY1) was secreted in >10-fold higher amounts than rAMY2 by heterologous expression in yeasts (Søgaard et al., 1990Go; Juge et al., 1993Go, 1996Go). Even the normally efficient host Pichia pastoris strain GS115, produced only 1 mg/l rAMY2 (Juge et al., 1996Go), i.e. an impractically low level for most structural, biochemical and biophysical characterizations. Moreover, site-directed mutagenesis has previously caused considerably reduced yields (Søgaard et al., 1993Go; Mori et al., 2002Go).

A variety of molecular biological techniques have been applied to overcome poor protein yields in heterologous expression. These include suppression of transgene silencing in transgenic plants, utilization of methylotrophic yeasts as host for eukaryotic protein expression, fusion with protein tags, co-expression of chaperones to support correct folding of target proteins, etc. (Olins and Lee, 1993Go; De Wilde et al., 2000Go; Gellissen, 2000Go). Furthermore, methodologies of directed evolution have proved to be efficient tools for producing recombinant proteins in heterologous hosts (Roodveldt et al., 2005Go). Because it is a general aim to obtain the natural form, relatively few cases used mutation of the protein as a way to improve the yield. In one such case, however, development of structure–expression level relationship mutants of the extracellular domain of p55 tumor necrosis factor receptor displayed on the yeast cell surface consisted in proline substitution that promoted correct disulfide bond formation for a neighboring cysteine (Schweickhardt et al., 2003Go). In another example, engineering of insulin precursor resulted in enhanced folding stability accompanied by improved secretory efficiency (Kjeldsen et al., 2002Go). Finally, enhanced thermodynamic stability, but not oxidative folding or refolding rates in vitro of BPTI (bovine pancreatic trypsin inhibitor) mutants, strongly correlated with increased secretory efficiency in S.cerevisiae (Kowalski et al., 1998aGo,bGo).

Production of chimeras of barley {alpha}-amylases in S.cerevisiae strain S150-2B earlier suggested that differences in the N-terminal sequence influenced secretory efficacy of rAMY1 and rAMY2, but that signal sequences were without effect (Juge et al., 1993Go). Thus the chimeras r1N21 and r1N26, in which AMY2 N-terminal segments of 21 and 26 residues were substituted by the equivalent AMY1 sequences, were expressed poorly as judged from halo size on starch plates, whereas replacement with 53, 67 or 90 residues AMY1 segments increasingly improved the secreted yields (Juge et al., 1993Go). Based on this finding, rational engineering of rAMY2 involved combinatorial substitution at the 10 non-conserved positions in His14–Gln49 by the corresponding AMY1 residues. The mutagenesis was performed using degenerate oligonucleotide gene shuffling (DOGS), which is highly suited for family shuffling of closely related genes and leads to few non-shuffled products (Gibbs et al., 2001Go). Starch plate screening of the resulting combinatorial expression library in S.cerevisiae identified rAMY2 variants with a good level of secreted activity. Access to increased amounts essentially of rAMY2 was the primary goal of this work, since it is a most critical requirement for future studies and development of AMY2 by using directed evolution and screening. The successful overall strategy includes upscaling of rAMY2 in P.pastoris, a host that secures highly efficient production (Juge et al., 1996Go), but is incompatible with expression screening of variant libraries on starch plates.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Strains, plasmids and proteins

S.cerevisiae strains S150-2B (MAT{alpha} leu2-3 leu2-112 his3-D trp1-289 ura3-52) and INVSc1 (MAT{alpha} leu2 his3-D trp1-289 ura3-52) and P.pastoris strain GS115 (his4) (Invitrogen) were used as hosts. The earlier constructed plasmids pBAL7 and pBAH15 produce rAMY1 and rAMY2, respectively, in S.cerevisiae under control of yeast PGK promoter (Søgaard et al., 1990Go). pHIL-D2{alpha}2 is a pHIL-D2 (Invitrogen) derivative for producing rAMY2 in P.pastoris (Juge et al., 1996Go). pPGKYES2amy1 and pPGKYES2amy2, harboring AMY1 and AMY2 genes with their own signal sequence downstream of the PGK promoter, were derived from the pYES2/CT shuttle vector (Invitrogen) (Kim et al., 2003Go). AMY2 wild-type (AMY2-2 form) and BASI were purified from kilned malt (cv. Alexis) (Ajandouz et al., 1992Go) and from barley seeds (cv. Piggy) (Abe et al., 1993Go), respectively.

Construction of a mutant library by degenerate oligonucleotide gene shuffling (DOGS) and homologous recombination in yeast

The procedure for the construction of mutant genes is summarized in Figure 1. The target mutations were performed by PCR using degenerate oligonucleotide primers (Table I), pPGKYES2amy2 as template and Super Taq plus DNA polymerase (HT Biotechnology). Some mutant-containing fragments were generated by a subsequent overlap extension PCR using the above primers and first PCR products as templates (Figure 1A). pPGKYES2amy2 (100 ng) was linearized by BamHI and SacI and introduced with the final PCR products (500 ng) in yeast strain INVSc1 in the presence of carrier DNA (80 µg, salmon sperm, denatured; Sigma) for combination of fragment ends in overlap regions by the natural yeast homologous recombination (Figure 1B) (Orr-Weaver et al., 1981Go; Orr-Weaver and Szostak, 1983Go). The target comprises 19 nucleotides that vary between the AMY1 and AMY2 gene sequences encoding His14–Gln44 (AMY2 numbering; Figure 2A). At five positions with silent mutations either the AMY2 cDNA or the preferred S.cerevisiae codon was applied in the degenerate primers (Table I). Each amino acid substitution was configured to occur at a rate of 10%, except for Phe21, Ile34 and Glu48. Thus an ideal minimum library should contain ≥3 x 107 colonies, i.e. 107 colonies for each library with Phe21, Ile34 or Glu48 substituted to Met, Val and Asn, respectively. This strategy was chosen because F21M and E48N need two base changes, which would have greatly increased the required library size. Moreover, since Ile34AMY2 was far from the other target residues, specific primers (KF3 and KF6) were designed for the I34V replacement (Table I; Figure 1A).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1. Schematic representations of the generation of mutated genes by using DOGS (A) and yeast in vivo recombination (B). Various lengths of PCR products were obtained by applying degenerate oligonucleotide primers (KF1–KF8 and Fusion 228C; see Table I) in all combinations. For certain oligonucleotide fragments, a second overlap extension PCR was performed to obtain longer overlap regions that increased the frequency of recombination in yeast. P-PGK, yeast pgk promoter; SS, AMY2 signal peptide coding sequence. The expression vector was linearized by combined BamHI and SacI digestion, hence no parental gene was retrieved in the DOGS library.

 

View this table:
[in this window]
[in a new window]
 
Table I. Degenerate oligonucleotide primers for construction of a DOGS library representing AMY1 vs AMY2 sequence differences in His14–Gln49

 


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 2. Partial cDNA and amino acid sequence alignment of AMY1 and AMY2 (A) and the selected 6 mutants (B). Bold letters represent positions where mutations were introduced by DOGS. Nucleotide numbering (n.t.) is from the start codon and amino acid numbering (a.a.) from the N-terminal residue of the mature protein. In (B), h and s indicate {alpha}-helix and ß-strand, respectively.

 
Selection of positive clones and sequence identification of rAMY2 mutants

Positive clones were picked from Ura selective plates, inoculated on YPD-starch plates (1% yeast extract, 2% peptone, 2% glucose, 2% agar, 1% starch) and incubated for 48 h at 37°C followed by activity staining with 1.3% I2–3% KI aqueous solution (Thomsen, 1983Go). Colonies forming larger haloes than S150-2B harboring pPGKYES2amy2 were collected. Plasmid DNA was isolated from cell lysates by using acid-washed glass beads (Sigma) and a QIAprep Spin Miniprep Kit (QIAGEN) according to the manufacturer's instructions and mutated positions were identified [ABI310 DNA sequencer; BigDye Terminator v3.1 Cycle Sequencing Kit (PE Biosystems)].

Growth profiles in S.cerevisiae and purification of rAMY2 variants

Single colonies of selected variants were inoculated in 50 ml of YPD (1.0% yeast extract, 2% peptone, 2% glucose) in 300 ml flasks and shaken at 300 r.p.m. for up to 168 h. Aliquots (1 ml) of culture medium were collected at time points and OD600 and the activity towards insoluble Blue Starch were measured. For the preparative scale, a single colony was inoculated in 100 ml of YPD, pre-cultivated at 30°C for 2 days and 10 ml were added to 2.5 l of YPD (in 5 l flask). After 3 days at 30°C, cells were removed by centrifugation (4200 r.p.m., 30 min). The supernatant was concentrated (0.5 l; Pellicon 2 TFF Systems; membrane pore size 0.22 µm; Millipore), made 5% saturated in ammonium sulfate and applied to ß-cyclodextrin–Sepharose ({phi} 1.6 x 2.0 cm) equilibrated with 20 mM sodium acetate, 25 mM CaCl2, pH 5.5 (buffer A), washed (buffer A containing 200 mM NaCl, 10 column volumes) and protein was eluted (buffer A containing 8.0 mg/l ß-cyclodextrin, 10 column volumes). A flow rate of 25 ml/h was used in all the above chromatographic steps. The eluate was concentrated (500 µl; Centriprep YM-30; Millipore; 2200 r.p.m.), diluted with 20 mM MES, pH 6.8, containing 1 mM CaCl2 (4.5 ml) and applied to a ResourceQ column (6 ml) pre-equilibrated with the same buffer and eluted with a 0–0.3 M NaCl linear gradient in the buffer (flow rate 1 ml/min) using an ÄKTAexplorer chromatograph (Amersham). Fractions containing enzyme were pooled and concentrated to 500 µl followed by buffer exchange for storage in 20 mM MES, 25 mM CaCl2, 0.02% (w/v) sodium azide, pH 6.8 (Centriprep YM-30, as above). The ion-exchange chromatography step was omitted if affinity chromatography gave a single component (SDS–PAGE). Protein concentration was calculated from amino acid contents of hydrolyzates (110°C, 24 h, 6 M HCl) of aliquots (0.2 nmol) on a Biochrom 20 Amino Acid Analyzer or estimated using (Gibson and Svensson, 1986Go). For SDS–PAGE enzyme (5–10 µg) was denatured with NuPAGE LDS Sample Buffer at 100°C for 5 min, loaded on NuPAGE 10% Bis-Tris Gel 1.0 mm, run at 200 V on an Xcell Sure Lock Mini-Cell and stained with a Colloidal Blue Staining kit according to the supplier's (Invitrogen) instructions.

Production of A42P and wild-type rAMY2 in P.pastoris

The single mutant A42P and wild-type rAMY2 were produced in the methylotrophic yeast P.pastoris strain GS115. pHIL-D2{alpha}2 (Juge et al., 1996Go) was used as a template for constructing the expression vectors. To eliminate unfavorable 5'- and 3'-untranslated regions in the template vector and to introduce the A42P mutation, oligonucleotides were amplified by PCR using the following primers: M6op1, 5'-TTTTTAGGAATTCCACCATGGCGAACAAACACTTG-3' (sense chain, outer primer); M6op2, 5'-TTTTTGAATTCATATTTTCTCCCATACGGCGTAG-3' (antisense chain, outer primer); M6fwd, 5'-CTCCCTCCGCCGTCGCAGTC-3' (sense chain, for mutation); and M6rev, 5'-GACTGCGACGGCGGAGGGAG-3' (antisense chain, for mutation) (EcoRI is singly underlined; A42P codon is doubly underlined; italics and bold indicate Kozak consensus sequence and start codon, respectively). The full-length PCR amplicons and pHIL-D2{alpha}2 were digested with EcoRI, combined with T4 DNA ligase (New England Biolabs) and transferred into E.coli TOP10 competent cells (Invitrogen). The resulting expression plasmids, pHIL-D2{alpha}2{Delta}UTR and pHIL-D2{alpha}2M6{Delta}UTR harboring wild-type and A42P rAMY2, respectively, were purified from positive clones screened on LB plates containing 100 µg/l ampicillin. Correct insert orientation was confirmed by restriction enzyme analysis using PvuI and the entire coding region was sequenced as described above. pHIL-D2{alpha}2{Delta}UTR and pHIL-D2{alpha}2M6{Delta}UTR were digested with NotI, introduced into P.pastoris strain GS115 by electroporation (0.2 cm cuvettes, 1.8 kV, 5 ms) and His+ Muts phenotypes were selected following the manufacturer's instruction. A42P and wild-type rAMY2 were produced by P.pastoris transformants (Juge et al., 1996Go), except that 0.5% (v/v) instead of 2% (v/v) methanol was used for induction and purified as described above. {alpha}-Amylase activity in the induction culture liquid (400 ml) was assayed using insoluble Blue Starch as substrate (Juge et al., 1996Go).

Enzyme activity assays

Insoluble Blue Starch Enzyme (1 nM) was added to 6.25 mg/l insoluble Blue Starch (customer preparation, Pharmacia) in 20 mM sodium acetate, 5 mM CaCl2, 0.5 mg/l BSA, pH 5.5 (4 ml) at 37°C. The reaction was stopped at 15 min with 0.5 M NaOH (1 ml) and centrifuged (13 000 r.p.m.; 5 min). The absorbance of the supernatant (300 µl, in duplicate) was measured in a microtiter plate at 620 nm (MRX II Absorbance Reader; DYNEX Technologies); 1 U was defined as the amount of enzyme that gave an increase of one absorbance unit during 15 min of reaction measured for the final (5 ml) mixture (Mori et al., 2002Go).

Amylose DP17 Hydrolysis of amylose DP17 (average degree of polymerization 17, Hayashibara Chemical Laboratories, Okayama, Japan) was measured after 10 min and at 10 substrate concentrations [0.001–1.0% (w/v)] using 0.4–1.0 nM {alpha}-amylase at 37°C in 20 mM sodium acetate (pH 5.5) containing 0.5 mg/l BSA. Released reducing sugar was determined by the copper bicinchoninate method using maltose as standard (McFeeters, 1980Go; Mori et al., 2002Go) and kcat and KM were calculated by fitting hydrolysis rates to the Michaelis–Menten equation (GraFit 3.02; Erithacus Software).

2-Chloro-4-nitrophenyl ß-D-maltoheptaoside (Cl-PNPG7) Initial rates of hydrolysis at 30°C were monitored for up to 8 min using 4–24 nM {alpha}-amylase and 0.25–10 mM Cl-PNPG7 (Granutest 3, Merck) in 100 µl of 50 mM phosphate buffer, pH 6.8, 50 mM KCl, 0.02% sodium azide, 17 U {alpha}-glucosidase from Escherichia coli and 0.26 U ß-glucosidase from almond (Mori et al., 2002Go). kcat and KM were determined as above.

pH and Ca2+ dependence of activity

Enzyme (1 nM) activity was measured essentially as before (Søgaard and Svensson, 1990Go) using insoluble Blue Starch as substrate (see above) in 20 mM sodium acetate, 1 mM CaCl2, 0.5 mg/l BSA (pH 3.0–6.0) and in 20 mM sodium phosphate, 1 mM CaCl2, 0.5 mg/l BSA (pH 6.5–8.0) for 15 min. The effect of CaCl2 (0–50 mM) on enzyme (1 nM) activity towards insoluble Blue Starch was measured as described above.

Stability at acidic pH and at elevated temperature

The stability of AMY2 wild-type and mutants (6–19 nM) during 10-120 min of incubation in 20 mM sodium citrate, 1 mM CaCl2, pH 3.5 at 37°C was assessed from residual activity towards insoluble Blue Starch (Juge et al., 1995Go). The thermostability was described by residual activity (as above) after 10 min of incubation at 10 different temperatures in the range 65–82°C (Jensen et al., 2003Go).

Inhibition by barley {alpha}-amylase/subtilisin inhibitor (BASI)

BASI (eight concentrations, 0–970 nM) and wild-type or rAMY2 mutants (10 nM) were incubated (40 mM Tris–HCl, 5 mM CaCl2, 0.5 mg/l BSA, pH 8.0) for 15 min at 30°C followed by assay of residual activity towards insoluble Blue Starch (Bønsager et al., 2003Go). The extent of inhibition was calculated from 100 x [1 – (Acti/Act0)], where Acti and Act0 represent {alpha}-amylase activity in the presence and in the absence of BASI.

Crystallization

A42P rAMY2 (2.58 mg/l) stock in 10 mM MES, 25 mM CaCl2, pH 6.8 was crystallized using hanging or sitting drops equilibrated against a reservoir solution containing 1.6 M ammonium sulfate, 0.1 M MES (pH 6.5) and 10% dioxane. The drops were composed of 1:1 protein stock and reservoir solution and left at 19°C. Crystals appeared after a few days growth to approximately 0.2 x 0.2 x 0.05 mm and resembling native AMY2 crystals (Svensson et al., 1987Go). To improve resolution, the crystals were soaked with acarbose added to a final concentration of 10 mM in the drop 20 h prior to data collection. Ethylene glycol was added to the drop to a final concentration of 15% as a cryoprotectant.

Diffraction collection, data processing and refinement

Diffraction data for A42P rAMY2 complexed with acarbose (A42P/aca) was collected on a MARCCD 165 detector at the ID14-3 beamline at the synchrotron ESRF (Grenoble). The cryoprotected crystal was kept at 100 K in a nitrogen stream during data collection and diffracted X-rays to 2.2 Å resolution. Processing of the data and integration of the diffracted intensities was done with the XDS package (Kabsch, 1993Go) and scaled with XSCALE (Diederichs et al., 2003Go). The space group was found to be trigonal (P3121) with a = b = 133.4 Å, c = 80.2 Å, {alpha} = ß = 90° and {gamma} = 120° essentially as for native AMY2 crystals (Kadziola et al., 1994Go). The structure was solved by molecular replacement using the CNS software (Brünger et al., 1998Go) and the structure of the native AMY2 (PDBid 1AMY at resolution 2.8 Å) as search model, giving rise to a single solution, using mutant data in the range 10–4 Å. After rigid body refinement simulated annealing was carried out (data between 50 and 2.2 Å resolution were withdrawn) with programs from the CNS software; model building and manual fitting were performed with the program TURBO-FRODO (Roussel and Cambillau, 1989Go). A test set was set aside including 10% of the reflections which was used to calculate R-free during the refinement. The R-factor and R-free for the final structure were 18.2% and 21.5%, respectively. Water molecules were added when electron densities were at least 1{sigma} (in the 2FoFc map) and 3{sigma} (in the Fo Fc map) with the possibility of forming appropriate hydrogen bonding and omitted if B-factors exceeded 60 Å2. At the end of the refinement, PROCHECK (Laskowski et al., 1993Go) was used to analyze the stereochemistry of the model.

Molecular graphics and simulational analysis

Molecular graphics were generated by using Swiss-PdbViewer (http://us.expasy.org/spdbv/) (Guex and Peitsch, 1997Go) and rendered by POV-Ray (http://www.povray.org/). Protein Data Bank (http://www.rcsb.org/pdb/) accessions 1HT6 and 1AMY were used for depicting AMY1 and AMY2 structures. Probable steric clashing and established hydrogen bonds in mutant proteins were assessed using Swiss-PdbViewer.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Screening of variants forming large haloes on starch plates

In the first screen 1.8 x 104 colonies were obtained on Ura selective plates and part of those were examined for secreted {alpha}-amylase activity by transfer to starch-plates. In S.cerevisiae strain INVSc1 six mutants (M1–M6) were identified of 843 clones to form large haloes (Figure 3A). Surprisingly, the halo size of rAMY2 in this strain exceeded that of rAMY1 (Figure 3A), which had yielded the larger haloes in the other host strain S150-2B (Figure 3B) (Søgaard and Svensson, 1990Go; Juge et al., 1993Go). The relative halo size of rAMY1 and rAMY2 was maintained with expression plasmids pBAH15 and pPGKYES2amy2 and M1–M6 produced larger haloes in S150-2B than rAMY2 wild-type (Figure 3B). Compared with rAMY2 M2, M4 and M5 gave larger, M1, M6 the same size and M3 slightly smaller halo in INVSc1 (Figure 3A) and after 71 h liquid culture M1–M6 produced 0.63–2.9 times the activity of wild-type consistent with the halo sizes (Figure 3A and C). For unidentified reasons such a correlation of activity in liquid culture and starch plate halo size was not found in S150-2B (Figure 3B and D). rAMY1 in INVSc1, in agreement with relative halo sizes, gave about 0.02 mg/l in liquid culture corresponding to about 15% of rAMY2 in this host, but up to 1.1 mg/l or 20-fold higher amount than rAMY2 in S150-2B. Based on the specific activity of purified rAMY2 of 5530 U/mg (this study), 0.16 mg/l was secreted by INVSc1, which is roughly three times more than by S150-2B.



View larger version (50K):
[in this window]
[in a new window]
 
Fig. 3. Comparison of halo size of rAMY1, rAMY2 and six large-halo forming rAMY2 variants [(A) and (B)] selected by screening using I2–KI staining of starch-containing YPD plates and {alpha}-amylase activity in liquid culture towards insoluble Blue Starch [(C )and (D)]. Construction of expression plasmids of AMY2 mutants in pPGKYES2 derived from the pYES2/CT shuttle vector (see Materials and methods). (A) Host strain INVSc1. pBAL7 and pBAH15 were introduced in this strain as control of secretion of rAMY1 and rAMY2, respectively. (B) Host strain S150-2B. pBAL7 and pPGKYES2amy1 encoded for rAMY1, pBAH15 and pPGKYES2amy2 encoded rAMY2. (C) Cell growth (dotted lines) showing no significant differences for the six mutants and {alpha}-amylase activity (full lines) in liquid culture with INVSc1 and in (D) with S-150-2B as host. Circles, rAMY1 encoded by pBAL7; triangles, rAMY2 encoded by pBAH15; squares, M1; diamonds, M2; x, M3; *, M4; –, M5; +, M6.

 
Structural implications of amino acid substitutions in selected gene shuffled rAMY2 variants

Substitutions in M1–M6 occurred chiefly in the sequence Pro42–Gln49 in the ß->{alpha}-loop 2 of the catalytic (ß/{alpha})8-barrel (Kadziola et al., 1994Go). The selected mutants M1, M2 (both A42P/A47S), M4 (F21M/Q44H) and M6 (A42P) comprised only four of the 10 designed replacements and substitution was not identified of His14AMY2, Asn15AMY2, Leu22AMY2 and Ile34AMY2, included in the DOGS. F4L and N218D were unwanted and probably due to PCR errors found in triple mutants M3 (F21M/Q44H/E48N) and M5 (A47S/E48N/Q49E) (Figure 2B), respectively, which therefore were not further characterized. M1 and M2 both encoded A42P/A47S, but M2 secreted higher amounts in solid and liquid cultures and M1 was not further characterized. Sequencing identified the Phe7 codon having C and T as the third base in M1 and M2, respectively. C was also found in wild-type and the five other selected mutants.

The six mutated residues are superimposed in AMY1 and AMY2 (Figure 4A) and shown in detail (Figure 4B–D). The A42P rAMY2 (Figure 4D) was structure determined. Phe21AMY2 in the first turn of {alpha}-helix 1 of the catalytic (ß/{alpha})8-barrel points into solvent and F21M probably does not alter function or stability. A47S (in M2) by computational analysis was found to form a new hydrogen bond to Gln49AMY2 N{varepsilon}2 or Ser45AMY2 O{gamma} (not shown). The corresponding Ser48AMY1 O{gamma} hydrogen bonds to Ser46AMY1 O{gamma} and Met53AMY1 N (Robert et al., 2003Go), supporting the formation of hydrogen bonds of A47S to Ser45AMY2 and Met52AMY2 (Figure 4B) probably stabilizing ß->{alpha}-loop 2. Eight Met53AMY1 mutants showed their role in substrate interaction at the high-affinity subsite –2 important in activity and specificity (Mori et al., 2002Go). Q44H (in M4) might retain a hydrogen bond to Arg55AMY2 N (Figure 4C). E48N (in M3 and M5, see above) represented Asn49AMY1 and Glu48AMY2, both exposed to solvent and forming no intramolecular hydrogen bonds (Figure 4B). AMY1 and AMY2 crystal structures suggested that changes in the M2, M4 and M6 in ß->{alpha}-loop 2 adjacent to domain B, conferring AMY1 and AMY2 isozyme characteristics (Rodenburg et al., 1994Go), had no critical effect on the backbone conformation. The structure of A42P (M6; Figure 4D) is described below.



View larger version (52K):
[in this window]
[in a new window]
 
Fig. 4. Stereo views highlighting amino acid residues replaced in the AMY2 mutants (AMY2 numbering). (A) Substituted residues, Phe21, Ala42, Gln44, Ala47, Glu48 and Gln49 are shown by wire frame representation in orange; the corresponding AMY1 residues are in blue. This color code applies to all panels. (B) Close-up of a hydrogen bond network related to M2 where Ser48AMY1 O{gamma} (corresponds to Ala47AMY2) forms two hydrogen bonds to Ser46AMY1 O{gamma} (Ser45AMY2) and Met53AMY1 N (Met52AMY2).(C) Superimposition of His45AMY1 and Gln44AMY2 related to Q44H in M3 and M4. Hydrogen bonds shown between His45AMY1 and Arg56AMY1 (Arg55AMY2) may form in the mutants. (D) Superposition of the structures of A42P rAMY2 (M6; pink), AMY1 and AMY2. The stereo picture shows residues around the mutation site; Leu39AMY2 (Leu40AMY1), Pro40AMY2 (Pro41AMY1), Pro41AMY2 (Pro42AMY1), Pro42AMY2 (Pro43AMY1), Ser43AMY2 (Ser44AMY1), Wat755A42P, Wat760A42P, Wat862A42P, Wat757AMY1, Wat760AMY1, Wat756AMY1 and Wat734AMY2. It is clear that the backbone of M6 occupies the same conformation as AMY1 and the shift in the backbone is due to the replacement of the alanine by a proline. This conformational shift leaves room for three new water molecules in M6, also present in the AMY1 structure. Furthermore, a water molecule previously found in AMY2 is absent in A42P, as in AMY1, because the space now is occupied by the proline.

 
Enzymatic and stability properties of AMY2 mutants

M2, M4, M6 and wild-type rAMY2 were secreted at 0.39, 0.29, 0.31 and 0.16 mg/l, respectively, as calculated from the specific activities towards insoluble Blue Starch. The M6 thus doubled the yield, which M2 further increased by 30%. The mutants showed slightly higher stability at pH 3.5 than AMY2 (Figure 5A). M4 was most stable and retained 12% activity after 30 min compared with AMY1 retaining 50% (Rodenburg et al., 2000Go) and thus acquired AMY1-like behavior. M4 Q44H was exposed to solvent and probably interacted with Arg55AMY2 (Figure 4C), as for the hydrogen bond between Gln44AMY2 O{varepsilon}1 (His45AMY1 N{delta}1) and Arg55AMY2 N (Arg56AMY1 N).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5. Comparison of stability and enzymatic properties of AMY2 (open circles) and mutants M2 (closed circles), M4 (closed triangles) and M6 (closed squares). (A) Stability at pH 3.5. (B) pH–activity profiles. (C) Inhibition of activity by BASI. (D) Effect of Ca2+ on activity. (E) Thermal stability of AMY2, M2 and M6.

 
M2, M4, M6 and wild-type AMY2 had very similar pH activity optima at pH 6.5–7.0 (Figure 5B). The sigmoid acid limb for M4 might stem from deprotonation of the introduced His44. These variants furthermore retained the sensitivity of AMY2 for BASI (Figure 5C; Table II). Hence these ß->{alpha}-loop 2 mutations did not elicit changes affecting the neighboring domain B that is part of the protein interface in AMY2/BASI (Vallée et al., 1998Go). The characteristic optimum activity of AMY2 around 10 mM Ca2+ was shared by the mutants (Figure 5D), also suggesting that no critical change occurred in and near domain B that binds all three structural Ca2+. M2 and M6, containing A42P, had essentially the AMY2 thermostability (Figure 5E).


View this table:
[in this window]
[in a new window]
 
Table II. Enzyme kinetic parameters, relative substrate specificity and inhibition by BASI of the selected AMY2 variants

 
Enzyme kinetic parameters for three different substrates, insoluble Blue Starch, a maltodextrin amylose DP17 and an oligosaccharide Cl-PNPG7, were essentially identical for M6 and wild-type AMY2, but deviated for M2 and M4 (Table II). Since M6 (A42P) maintained activity on both amylose DP17 and Cl-PNPG7, the loss for M2 (A42P/A47S) in catalytic efficiency of 30–50% due to reduced kcat and increased KM, respectively, was ascribed to A47S at subsite –3, which may rigidify ß->{alpha}-loop 2 by an extra hydrogen bond and perturb the important Met52AMY2 at subsite –2 (Mori et al., 2002Go). M4 (F21M/Q44H) differed from AMY2 on all substrates and preferred amylose for Cl-PNPG7 as seen from KM and kcat (Table II). Whereas F21M is far from the active site (Figure 4A), Q44H may interact with substrate or have a second shell contact and confer M4 AMY1-like enzymatic properties.

Production of A42P (M6) and wild-type rAMY2 in P.pastoris

Expression profiles of A42P and wild-type rAMY2 were examined for P.pastoris transformants showing the highest activity in the induction medium. A42P was stably secreted after 48 h of cultivation at a level of 60 mg/l as calculated from the specific activity towards insoluble Blue Starch and yielded a single band in SDS–PAGE (not shown) after purification by affinity chromatography on ß-cyclodextrin–Sepharose. In the same expression system the maximum yield found for rAMY2 wild-type was 3 mg/l. Thus elimination of the 5'- and 3'-untranslated regions (~800 bp) raised the secretory expression of rAMY2 by 3-fold compared with previously (Juge et al., 1996Go).

Three-dimensional structure of A42P rAMY2

The structures of A42P rAMY2 produced in P.pastoris and native AMY2 (Kadziola et al., 1994Go) were essentially identical. In A42P rAMY2 (for final refinement statistics, see Table III), however, the loop between Tyr378 and Gly387 was not as well defined as the rest of the structure (to be described in detail elsewhere). The mutant Pro42, located in the (ß/{alpha})8-barrel (domain A) in the beginning of loop 2 between strand A-ß2 and helix A-{alpha}2 (Kadziola et al., 1994Go), was easily built into the electron density (Figure 6). Superposition of A42P rAMY2, AMY2 (PDB 1AMY), AMY2 in complex with acarbose (AMY2/aca, PDB 1BG9), AMY1 (PDB 1HT6) and AMY1 in complex with acarbose (PDB 1RPK) reveal structural differences (Figure 4D). Superposition on the basis of C{alpha} of Trp18, Asp58, Lys63, Ala84, Ile88 and Ile171, near A42P, showed that the mutation slightly shifted the backbone compared with AMY2 and AMY2/aca. Hence the region comprising residues 39–41 moved ~0.6 Å, but Pro42 C{alpha} remained at the same position as in AMY2 and AMY2/aca (Figure 4D). Remarkably, the A42P AMY2 loop adopted the same conformation as in AMY1 and AMY1/aca, making room for three additional water molecules (Wat755, Wat760 and Wat862), also present in AMY1 (Wat757AMY1, Wat760AMY1, Wat756AMY1) (Robert, et al., 2003Go, 2005Go). Wat755 hydrogen bonded to Ser62 O{gamma} (2.8 Å), Ser43 O (2.7 Å), Wat606 (2.8 Å) and Pro41 O (2.6 Å), Wat760 to Ser62 O{gamma} (3.5 Å), Leu59 O (3.3 Å), Gly65 O (3.1 Å) and Tyr19 OH (2.8), while Wat862 hydrogen bonded to Ser43 O (2.9 Å), Wat615 (2.8 Å), Gly50 O (2.8 Å) and had more distant interactions with Wat755 (3.9 Å) and Gln44 O (3.8 Å). Wat755 and Wat862 were located next to each other under the A42P rAMY2 tri-proline loop, whereas Wat760 was found on the other side of this loop (Figure 4D). Furthermore, one water (Wat734AMY2), present in AMY2 structures (Kadziola et al., 1994Go, 1998Go), was absent in A42P rAMY2, AMY1 and AMY1/aca. The imino ring of Pro42 occupied the space which in native AMY2 accommodated Wat734. Structural differences described above between A42P rAMY2, AMY2 and AMY1 facilitated only very small shifts in the surrounding protein backbone and side chains, without significant changes in the overall structure. Consequently, the new conformation in A42P rAMY2 represents no clear structural reason for the stabilization, hence thermodynamic stabilization of ß->{alpha}-loop 2 is proposed to explain the improved heterologous expression and production of A42P rAMY2.


View this table:
[in this window]
[in a new window]
 
Table III. Data collection and refinement statistics for rAMY2 mutant A42P complexed with acarbose

 


View larger version (59K):
[in this window]
[in a new window]
 
Fig. 6. Electron density maps showing the mutation A42P in M6. The electron density shown before (A) and after (B) the mutation was included in the refinement. 2FoFc electron density was contoured at 1{sigma} (presented in blue) and positive FoFc electron density is contoured at 3{sigma} (presented in green).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Numerous studies have dealt with the characterization of barley {alpha}-amylase AMY1, AMY2 and corresponding recombinant wild-type and mutant proteins. Nevertheless, the poor secretory expression of rAMY2 remained problematic and hampered mutational analyses of structure–function relationships or engineering of AMY2, which is naturally the major isozyme. Important factors in improving the amount of secreted recombinant protein by heterologous hosts include (i) host codon usage optimization, (ii) suitability of signal peptides, (iii) tolerance against host-producing proteases, (iv) stabilization of intermediate state of pre-mature protein and (v) stability and solubility of the mature protein. In the present study, optimized codon usage was examined for the sequence encoding the N-terminal target region of AMY2 (Figure 2A, Table I) identified by previous analyses of AMY1/AMY2 chimeras (Juge et al., 1993Go). The combinatorial approach using DOGS in conjunction with yeast in vivo recombination resulted in three rAMY2 variants, M2 (A42P/A47S), M4 (F21M/Q44H) and M6 (A42P), with improved secretory expression of 0.29–0.39 mg/l in S.cerevisiae strain INVSc1. In comparison, rAMY1 was obtained in highest yield of 1.1 mg/l in strain S150-2B (Søgaard et al., 1990Go). The mutant M6 was virtually identical with AMY2 wild-type in stability and enzymatic properties and was secreted from P.pastoris in 20-fold higher amounts than rAMY2, allowing mutational structure–function relationship investigations and protein engineering.

Codon bias is common in most species and in yeast a direct relationship was reported between codon usage and expression level (Bennetzen and Hall, 1982Go; Fuglsang, 2003Go). The Phe7 codon in M1 and M2, representing identical protein variants, had C and T as third base, respectively, presumably causing the 2-fold higher yield of M2. Even though UUU is slightly more frequent than UUC in S.cerevisiae (Codon Usage Database, Kazusa DNA Research Institute; http://www.kazusa.or.jp/codon/), U seemed less favorable here, since wild-type and the five other selected mutants have C. Furthermore, sequencing of linearized pPGKYES2amy2 lacking the N-terminal encoding region between the BamHI and SacI sites and integrated with DOGS PCR products by homologous recombination (Figure 1A and B), had 3–15 AMY2-encoding silent mutations adapted to codon usage preferred by S.cerevisiae (Table I, doubly underlined), but the corresponding colonies gave similar or smaller halo size on starch plates compared with wild-type, indicating that mostly the protein sequence was more important than codon usage for the level of secreted rAMY2. This was further supported by the exceptionally high yield of 60 mg/l A42P rAMY2 secreted without optimized codons by P.pastoris strain GS115, compared with 3 mg/l of wild-type rAMY2. Still, one cannot exclude that the mutation G to C encoding A42P is preferred by the yeast hosts increasing the production of recombinant protein. The impact of the signal sequence on the yield was not addressed, because secretion of rAMY2 previously did not increase after substitution with the AMY1 signal sequence (Juge et al., 1993Go).

Ala42AMY2 is buried in the catalytic domain and accommodation of proline at this position did not critically perturb the local conformation. Although Ala42AMY2 C{alpha} within 9 Å is surrounded by the hydrophobic Leu39, Pro40, Pro41, Gly50, Pro53, Leu56, Leu59, Ala61, Gly65, Leu70, Ile74, Ala86, Ile88, Val89, Trp163, Trp166, Leu167, Ile171 and Phe173, this was too distant to form a hydrophobic cluster. These hydrophobic residues are conserved in AMY1 and AMY2 and superimposed well except for Leu59AMY2 (Ile60AMY1) and Ile171AMY2 (Leu172AMY1). A unique di-trans-Pro sequence preceded both Ala42AMY2 and Pro43AMY1 (Figure 4D). Effect of proline engineering was examined in oligo-1,6-glucosidase from Bacillus cereus for which proline introduced at the second position of the ß-turns and at N-caps of {alpha}-helices enhanced thermostability (Watanabe et al., 1991Go). ß-Turns consisting of four consecutive residues are classified in nine types (I, II, VIII, I', II', VIa1, VIa2, VIb, IV) based on dihedral angles ({phi}, {psi}) at the second and third positions and the distance between C{alpha} of the first and the fourth residue (within 7 Å) (Guruprasad et al., 2000Go). Proline is generally favored at the second position and is least frequent at the third position in a ß-turn. Among type VI ß-turns, however, proline was observed at the third position supposed to be in two overlapping ß-turns sharing part of the constituent residues (Hutchinson and Thornton, 1994Go). Furthermore, a number of multiple ß-turns with proline in the third position were found in silico, showing distinct preferences of the dihedral angles in Ramachandran plots (Guruprasad et al., 2000Go). The characteristic tri-trans-Pro structures, including Pro43AMY1 and A42PM6, extend ~6 Å (from the first Pro C{alpha} to the third Pro C{alpha}) towards the molecular surface and do not form a ß-turn (Figure 4D). The proline substitution, however, most likely stabilizes the turn conformation to have constraints on the main-chain dihedral angles restricting rotation of the C{alpha} and N bond. It is speculated that Pro41AMY1 and Pro40AMY2 (Figure 2A) at the first position of the di- or tri-trans-proline sequence forming a turn at the start of ß->{alpha}-loop 2 might break the extension of ß-strand 2, hence facilitating folding of ß->{alpha}-loop 2. The hydrogen bonds Pro41 O–Pro43 N and Pro40 O–Ala42 N were observed in AMY1 and AMY2. Moreover, lack of steric hindrance between Pro42 in M6 and the surrounding residues might facilitate a more rigid hydrophobic packing in this area compared with wild-type rAMY2. It seems, therefore, that A42PAMY2 stabilizes ß->{alpha}-loop 2 thermodynamically. A strong connection between thermodynamic stability and protein yield was found previously by mutational analysis of the recombinant 6.5 kDa bovine pancreatic trypsin inhibitor produced in S.cerevisiae (Kowalski et al., 1998bGo) that folds and unfolds reversibly. Barley {alpha}-amylase is much larger (45 kDa) and, because it denatures irreversibly (Jensen et al., 2003Go), it is difficult to characterize and hence to correlate thermodynamic stability properties with expression level. The stability of barley {alpha}-amylases furthermore depends on three structural calcium ions (Kadziola et al., 1994Go, Robert et al., 2002Go, 2003Go) and numerous non-covalent interactions between the catalytic domain A and the small B and C domains. Previously immunoblot analysis revealed the major part of rAMY2 to accumulate intracellularly in S150-2B in contrast to rAMY1 (Juge et al., 1993Go). Presumably instability of either folding intermediates or apo-forms of rAMY2 causes the formation of off-pathway structures (Roodveldt et al., 2005Go). In the case of A42P rAMY2, the introduced proline may promote the correct folding of the native conformation by either destabilizing non-native structures or raising the kinetic barrier to their formation (Wigley et al., 2002Go).

In summary, the single substitution A42P in AMY2 enhanced the yield 20-fold of recombinant enzyme secreted by P.pastoris without changing the enzymatic and stability properties. This confirmed the hypothesis ascribing the poor yield of rAMY2 in various yeast hosts to the N-terminal region. Two probable factors for the enhanced secretion of A42P rAMY2 emerged: (i) stabilization of mature and/or intermediate form owing to the proline substitution and (ii) conversion of G to C at A42P, which perhaps improves the protein translation. To our knowledge, this is the first case of rational gene family shuffling used successfully to conduct a combinatorial screening to identify enhanced secretion by yeast of an enzyme variant with maintained wild-type characteristics. The achieved A42P rAMY2 is suitable for development of stability and enzymatic properties through directed evolution strategies in conjunction with expression screening and the exceptionally successful expression of A42P in P.pastoris secures a basis for future mutational and structural analyses in barley {alpha}-amylases. This is a very important example on yields of recombinant protein amplified in P.pastoris compared with modest albeit significant improvement identified by screening a library of variants in S.cerevisiae.


    Notes
 
3 Present address: Meat and Milk Hygiene, Graduate School Course of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-Sen 11-Banchi, Inada-Cho, Obihiro, Hokkaido 080-8555, Japan Back


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Sidsel Ehlers is gratefully acknowledged for expert technical assistance and Lone Sørensen for amino acid analyses. Drs Kirsten Bojsen, Sophie Bozonnet and Maher Abou Hachem are thanked for stimulating discussions on gene shuffling and stability of proteins. Dr Tae-jip Kim is thanked for a DOGS primer and expression vectors for S.cerevisiae strain INVSc1. This project was supported by grant QLK3-CT-2001-00149 to the EU FP 5 project ‘Combinatorial engineering of glycoside hydrolases from the {alpha}-amylase superfamily’ (CEGLYC) and by the Danish Natural Science Research Council.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Abe,J., Sidenius,U. and Svensson,B. (1993) Biochem. J., 293, 151–155.[ISI][Medline]

Ajandouz,E.H., Abe,J., Svensson,B. and Marchis-Mouren,G. (1992) Biochim. Biophys. Acta, 1159, 193–202.[ISI][Medline]

Bak-Jensen,K.S., André,G., Gottschalk,T.E., Paës,G., Tran,V. and Svensson,B. (2004) J. Biol. Chem., 279, 10093–10102.[Abstract/Free Full Text]

Bennetzen,J.L. and Hall,B.D. (1982) J. Biol. Chem., 257, 3026–3031.[Abstract/Free Full Text]

Brünger,A.T. et al. (1998) Acta Crystallogr. D, 54, 905–921.[CrossRef][ISI][Medline]

Bønsager,B.C., Prætorius-Ibba,M., Nielsen,P.K. and Svensson,B. (2003) Protein Expr. Purif., 30, 185–193.[CrossRef][ISI][Medline]

De Wilde,C., Van Houdt,H., De Buck,S., Angenon,G., De Jaeger,G. and Depicker,A. (2000) Plant Mol. Biol., 43, 347–359.[CrossRef][ISI][Medline]

Diederichs,K. and Karplus,P.A. (1997) Nat. Struct. Biol., 4, 269–275.[CrossRef][ISI][Medline]

Diederichs,K., McSweeney,S. and Ravelli,R.B.G. (2003) Acta Crystallogr. D, 59, 903–909.[CrossRef][ISI][Medline]

Fuglsang,A. (2003) Biochem. Biophys. Res. Commun., 312, 285–291.[CrossRef][ISI][Medline]

Gellissen,G. (2000) Appl. Microbiol. Biotechnol., 54, 741–750.[CrossRef][ISI][Medline]

Gibbs,M.D., Nevalainen,K.M. and Bergquist,P.L. (2001) Gene, 271, 13–20.[CrossRef][ISI][Medline]

Gibson,R. and Svensson,B. (1986) Carlsberg Res. Commun., 51, 295–308.[ISI]

Guex,N. and Peitsch,M.C. (1997) Electrophoresis, 18, 2714–2723.[ISI][Medline]

Guruprasad,K., Pavan,M.N., Rajkumar,S. and Swaminathan,S. (2000) Curr. Sci., 79, 992–994.[ISI]

Hutchinson,E.G. and Thornton,J.M. (1994) Protein Sci., 3, 2207–2216.[Abstract/Free Full Text]

Jensen,M.T., Gottschalk,T.E. and Svensson,B. (2003) J. Cereal Sci., 38, 289–300.[CrossRef][ISI]

Juge,N., Søgaard,M., Chaix,J.-C., Martin-Eauclaire,M.F., Svensson,B., Marchis-Mouren,G. and Guo,X.-J. (1993) Gene, 130, 159–166.[CrossRef][ISI][Medline]

Juge,N., Rodenburg,K.W., Guo,X.-J., Chaix,J.-C. and Svensson,B. (1995) FEBS Lett., 363, 299–303.[CrossRef][ISI][Medline]

Juge,N., Andersen,J.S., Tull,D., Roepstorff,P. and Svensson,B. (1996) Protein Expr. Purif., 8, 204–214.[CrossRef][ISI][Medline]

Kabsch,W. (1993) J. Appl. Crystallogr., 26, 795–800.[CrossRef][ISI]

Kadziola,A., Abe,J., Svensson,B. and Haser,R. (1994) J. Mol. Biol., 239, 104–121.[CrossRef][ISI][Medline]

Kadziola,A., Søgaard,M., Svensson,B. and Haser,R. (1998) J. Mol. Biol., 278, 205–217.[CrossRef][ISI][Medline]

Kim,T.-J., Bozonnet,S., Nielsen,P.K. and Svensson,B. (2003) In: Carbohydrate: Enzymes and Food Functionality, Proceedings 2003 Agricultural Biotechnology Symposium (Seoul National University), pp. 9–17.

Kjeldsen,T., Ludvigsen,S., Diers,I., Balschmidt,P., Sørensen,A.R. and Kaarsholm,N.C. (2002) J. Biol. Chem., 277, 18245–18248.[Abstract/Free Full Text]

Kowalski,J.M., Parekh,R.N., Mao,J. and Wittrup,K.D. (1998a) J. Biol. Chem., 273, 19453–19458.[Abstract/Free Full Text]

Kowalski,J.M., Parekh,R.N. and Wittrup,K.D. (1998b) Biochemistry, 37, 1264–1273.[CrossRef][ISI][Medline]

Laskowski,R.A., MacArthur,M.W., Moss,D.S. and Thornton,J.M. (1993) J. Appl. Crystallogr., 26, 283–291.[CrossRef][ISI]

McFeeters,R.F. (1980) Anal. Biochem., 103, 302–306.[CrossRef][ISI][Medline]

Mori,H., Bak-Jensen,K.S., Gottschalk,T.E., Motawia,M.S., Damager,I., Møller,B.L. and Svensson,B. (2001) Eur. J. Biochem., 268, 6545–6558.[Abstract/Free Full Text]

Mori,H., Bak-Jensen,K.S. and Svensson,B. (2002) Eur. J. Biochem., 269, 5377–5390.[Abstract/Free Full Text]

Mundy,J., Svendsen,I. and Hejgaard,J. (1983) Carlsberg Res. Commun., 48, 81–90.[ISI]

Olins,P.O. and Lee,S.C. (1993) Curr. Opin. Biotechnol., 4, 520–525.[CrossRef][Medline]

Orr-Weaver,T.L., Szostak,J.W. and Rothstein,R.J. (1981) Proc. Natl Acad. Sci. USA, 78, 6354–6358.[Abstract/Free Full Text]

Orr-Weaver,T.L. and Szostak,J.W. (1983) Proc. Natl Acad. Sci. USA, 80, 4417–4421.[Abstract/Free Full Text]

Robert,X., Haser,R., Svensson,B. and Aghajari,N. (2002) Biologia (Bratislava), 11, 59–70.

Robert,X., Haser,R., Gottschalk,T.E., Ratajczak,F., Driguez,H., Svensson,B. and Aghajari,N. (2003) Structure, 11, 973–984.[CrossRef][ISI][Medline]

Robert,X., Haser,R., Mori,H., Svensson,B. and Aghajari,N. (2005) J. Biol. Chem., in press.

Rodenburg,K.W., Juge,N., Guo,X.-J., Søgaard,M., Chaix,J.-C. and Svensson,B. (1994) Eur. J. Biochem., 221, 277–284.[Abstract]

Rodenburg,K.W., Vallée,F., Juge,N., Aghajari,N., Guo,X.-J., Haser,R. and Svensson,B. (2000) Eur. J. Biochem., 267, 1019–1029.[Abstract/Free Full Text]

Roodveldt,C., Aharoni,A. and Tawfik,D.S. (2005) Curr. Opin. Struct. Biol., 15, 50–56.[CrossRef][ISI][Medline]

Roussel,A. and Cambillau,C. (1989) TURBO-FRODO. Silicon Graphics, Mountain View, CA.

Schweickhardt,R.L., Jiang,X., Garone,L.M. and Brondyk,W.H. (2003) J. Biol. Chem., 278, 28961–28967.[Abstract/Free Full Text]

Søgaard,M. and Svensson,B. (1990) Gene, 94, 173–179.[CrossRef][ISI][Medline]

Søgaard,M., Olsen,F.L. and Svensson,B. (1991) Proc. Natl Acad. Sci. USA, 88, 8140–8144.[Abstract/Free Full Text]

Søgaard,M., Kadziola,A., Haser,R. and Svensson,B. (1993) J. Biol. Chem., 268, 22480–22484.[Abstract/Free Full Text]

Svensson,B., Gibson,R.M., Haser,R. and Astier., J.P. (1987) J. Biol. Chem., 28, 1368–13684.

Thomsen,K.K. (1983) Carlsberg Res. Commun., 48, 545–555.[ISI]

Vallée,F., Kadziola,A., Bourne,Y., Juy,M., Rodenburg,K.W., Svensson,B. and Haser,R. (1998) Structure, 15, 649–659.[CrossRef]

Watanabe,K., Chishiro,K., Kitamura,K. and Suzuki,Y. (1991) J. Biol. Chem., 266, 24287–24294.[Abstract/Free Full Text]

Wigley,W.C., Corboy,M.J., Cutler,T.D., Thibodeau,P.H., Oldan,J., Lee,M.G., Rizo,J., Hunt,J.F. and Thomas,P.J. (2002) Nat. Struct. Biol., 9, 381–388.[ISI][Medline]

Received April 27, 2005; revised August 5, 2005; accepted August 5, 2005.

Edited by Bauke Dijkstra





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
18/11/515    most recent
gzi057v1
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
Search for citing articles in:
ISI Web of Science (1)
Request Permissions
Google Scholar
Articles by Fukuda, K.
Articles by Svensson, B.
PubMed
PubMed Citation
Articles by Fukuda, K.
Articles by Svensson, B.