Production of Functional Lectin in<i>Pichia pastoris</i>Directed by Cloned cDNA from<i>Aleuria aurantia</i>

Amano, Koh; Takase, Mitsuyo; Andõ, Akira; Nagata, Yoshiho

doi:10.1271/bbb.67.2277

Cited by 14 publications

(9 citation statements)

References 13 publications

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“…The lectin was recently crystallized and each monomer was shown to have a sixfold β-propeller structure with five binding sites for L-fucose [8,14]. The slight structural differences at the five binding sites as well as the results from site specific mutagenesis studies indicated that the five possible binding sites for fucose differ in affinities towards fucose [8,14,15]. Site 2 and 4 have the highest affinities towards fucose, site 1 have medium affinity whereas site 3 and 5 bind fucose with the weaker affinities [8,14].…”

Section: Introductionmentioning

confidence: 99%

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Detection of a high affinity binding site in recombinant Aleuria aurantia lectin

Olausson¹,

Tibell²,

Jonsson³

et al. 2008

Glycoconj J

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Lectins are carbohydrate binding proteins that are involved in many recognition events at molecular and cellular levels. Lectin-oligosaccharide interactions are generally considered to be of weak affinity, however some mushroom lectins have unusually high binding affinity towards oligosaccharides with K (d) values in the micromolar range. This would make mushroom lectins ideal candidates to study protein-carbohydrate interactions. In the present study we investigated the properties of a recombinant form of the mushroom lectin Aleuria aurantia (AAL). AAL is a fucose-binding lectin composed of two identical 312-amino acid subunits. Each subunit contains five binding sites for fucose. We found that one of the binding sites in rAAL had unusually high affinities towards fucose and fucose-containing oligosaccharides with K (d) values in the nanomolar range. This site could bind to oligosaccharides with fucose linked alpha1-2, alpha1-3 or alpha1-4, but in contrast to the other binding sites in AAL it could not bind oligosaccharides with alpha1-6 linked fucose. This binding site is not detected in native AAL (nAAL) one possible explanation may be that this site is blocked with free fucose in nAAL. Recombinant AAL was produced in E. coli as a His-tagged protein, and purified in a one-step procedure. The resulting protein was analyzed by electrophoresis, enzyme-linked lectin assay and circular dichroism spectroscopy, and compared to nAAL. Binding properties were measured using tryptophan fluorescence and surface plasmon resonance. Removal of the His-tag did not alter the binding properties of recombinant AAL in the enzyme-linked lectin assay. Our study forms a basis for understanding the AAL-oligosaccharide interaction and for using molecular techniques to design lectins with novel specificities and high binding affinities towards oligosaccharides.

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Section: Introductionmentioning

confidence: 99%

“…The recombinant forms of AAL have been shown to retain their agglutinating properties [6,15]. However, there have been no previous studies that specifically analyze the specificity and affinity of recombinant AAL (rAAL).…”

Section: Introductionmentioning

confidence: 99%

Detection of a high affinity binding site in recombinant Aleuria aurantia lectin

Olausson¹,

Tibell²,

Jonsson³

et al. 2008

Glycoconj J

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“…One of the transformants was cultured in a 3-liter table-top glass fermenter (Model TS-M; Takasugi Seisakusho, Tokyo) using basal salts as the medium with the addition of methanol as a carbon source and an inducer for gene expression as reported. 8) Since PCL-M is known to have an affinity toward porcine stomach mucin (PSM), 6) it was purified by a batch-wise affinity chromatography using PSM as a ligand. PSM was coupled with N-hydroxysuccinimideactivated Sepharose (Amersham Biosciences).…”

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confidence: 99%

“…Tel/Fax: +81-47-308-8869; E-mail: ynagata@faculty.chiba-u.jp Abbreviations: PCL-M, mycelial aggregate-specific lectin of Pleurotus cornucopiae; PSM, porcine stomach mucin; TBS-Ca, Tris-buffered saline containing Ca 2þ ting as reported. 8) Protein contents were estimated by Bradford's method. 9) The expression product was soluble in TBS-Ca, and after abolishing EDTA, it showed hemagglutinating activity toward rabbit red blood cells.…”

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confidence: 99%

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Properties of Mycelial Aggregate-specific Lectin ofPleurotus cornucopiaeProduced inPichia pastoris

Sumisa

Iijima

Andõ

et al. 2004

Bioscience, Biotechnology, and Biochemistry

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cDNA of a mycelial aggregate-specific lectin of Pleurotus cornucopiae was expressed in Pichia pastoris, and the expression product was purified and characterized. The product was functional, and the hemagglutinating activity was inhibited most strongly by the addition of N-acetyl-D-galactosamine as was the native lectin. The native lectin is a glycoprotein having five glycosylation recognition signals, and the expression product showed slightly larger molecular mass than that of the native one due to further glycosylation.

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Mushroom Lectins

Goldstein

Winter

2007

Comprehensive Glycoscience

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Production of Functional Lectin inPichia pastorisDirected by Cloned cDNA fromAleuria aurantia

Cited by 14 publications

References 13 publications

Detection of a high affinity binding site in recombinant Aleuria aurantia lectin

Detection of a high affinity binding site in recombinant Aleuria aurantia lectin

Properties of Mycelial Aggregate-specific Lectin ofPleurotus cornucopiaeProduced inPichia pastoris

Mushroom Lectins

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