Expression of frutalin, an alpha-D-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris.

Frutalin is an alpha-D-galactose-binding lectin expressed in breadfruit seeds. Its isolation from plant is time-consuming and results in a heterogeneous mixture of different lectin isoforms. In order to improve and facilitate the availability of the breadfruit lectin, we cloned an optimised codifying frutalin mature sequence into the pPICZalphaA expression vector. This expression vector, designed for protein expression in the methylotrophic yeast Pichia pastoris, contains the Saccharomyces alpha-factor preprosequence to direct recombinant proteins into the secretory pathway. Soluble recombinant frutalin was detected in the culture supernatants and recognised by native frutalin antibody. Approximately 18-20 mg of recombinant lectin per litre medium was obtained from a typical small scale methanol-induced culture purified by size-exclusion chromatography. SDS-PAGE and Edman degradation analysis revealed that frutalin was expressed as a single chain protein since the four amino-acid linker peptide "T-S-S-N", which connects alpha and beta chains, was not cleaved. In addition, incomplete processing of the signal sequence resulted in recombinant frutalin with one Glu-Ala N-terminal repeat derived from the alpha-factor prosequence. Endoglycosidase treatment and SDS-PAGE analysis revealed that the recombinant frutalin was partly N-glycosylated. Further characterisation of the recombinant lectin revealed that it specifically binds to the monosaccharide Me-alpha-galactose presenting, nevertheless, lesser affinity than the native frutalin. Recombinant frutalin eluted from a size-exclusion chromatography column with a molecular mass of about 62-64 kDa, suggesting a tetrameric structure, however it did not agglutinate rabbit erythrocytes as native frutalin does. This work shows that the galactose-binding jacalin-related lectins four amino-acid linker peptide "T-S-S-N" does not undergo any proteolytic cleavage in the yeast P. pastoris and also that linker cleavage might not be essential for lectin sugar specificity.

[1]  F. Sumisa,et al.  Properties of Mycelial Aggregate-specific Lectin of Pleurotus cornucopiae Produced in Pichia pastoris , 2004, Bioscience, biotechnology, and biochemistry.

[2]  A. Monteiro-Moreira,et al.  Involvement of phosphatidylinositol-3 kinase-Akt and nuclear factor kappa-B pathways in the effect of frutalin on human lymphocyte. , 2006, International immunopharmacology.

[3]  C. Woloshuk,et al.  Cloning and functional expression of the gene encoding an inhibitor against Aspergillus flavus α-amylase, a novel seed lectin from Lablab purpureus (Dolichos lablab) , 2007, Plant Cell Reports.

[4]  N. Sharon,et al.  The binding of oligosaccharides containing N-acetylglucosamine and N-acetylmuramic acid to lysozyme. The specificity of binding subsites. , 1967, The Journal of biological chemistry.

[5]  L. Harvey,et al.  Heterologous protein production using the Pichia pastoris expression system , 2005, Yeast.

[6]  H. Yang,et al.  Isolation and characterization of cDNA clones encoding jacalin isolectins. , 1993, The Journal of biological chemistry.

[7]  E. V. Van Damme,et al.  The galactose‐binding and mannose‐binding jacalin‐related lectins are located in different sub‐cellular compartments , 2000, FEBS letters.

[8]  Y. Nagata,et al.  Production of fruiting-body lectins of Pleurotus cornucopiae in methylotrophic yeast Pichia pastoris. , 2003, Journal of bioscience and bioengineering.

[9]  M. I. Khan,et al.  Studies on recombinant single chain Jacalin lectin reveal reduced affinity for saccharides despite normal folding like native Jacalin , 2004, Protein science : a publication of the Protein Society.

[10]  M. Haine,et al.  Van Damme A. , 1986 .

[11]  P. Aucouturier,et al.  Structural and functional similarities of breadfruit seed lectin and jacalin. , 1990, Molecular immunology.

[12]  Unfolding and refolding studies of frutalin, a tetrameric D-galactose binding lectin. , 2001, European journal of biochemistry.

[13]  Ernst Kleinert,et al.  The Size of Г , 2000 .

[14]  C. Castelo-Branco,et al.  Isolation and partial characterization of a lectin from Artocarpus incisa L. seeds. , 1998, Phytochemistry.

[15]  J. Gatehouse,et al.  Functional phytohemagglutinin (PHA) and Galanthus nivalis agglutinin (GNA) expressed in Pichia pastoris correct N-terminal processing and secretion of heterologous proteins expressed using the PHA-E signal peptide. , 1999, European journal of biochemistry.

[16]  P. Proost,et al.  Two Distinct Jacalin-Related Lectins with a Different Specificity and Subcellular Location Are Major Vegetative Storage Proteins in the Bark of the Black Mulberry Tree1 , 2002, Plant Physiology.

[17]  A. Surolia,et al.  Structural basis of the carbohydrate specificities of jacalin: an X-ray and modeling study. , 2003, Journal of molecular biology.

[18]  A. Monteiro-Moreira,et al.  Frutalin, a galactose-binding lectin, induces chemotaxis and rearrangement of actin cytoskeleton in human neutrophils: involvement of tyrosine kinase and phosphoinositide 3-kinase. , 2005, Toxicology and applied pharmacology.

[19]  M. Taylor,et al.  Large-scale production, purification, and characterisation of recombinant Phaseolus vulgaris phytohemagglutinin E-form expressed in the methylotrophic yeast Pichia pastoris. , 2002, Protein expression and purification.

[20]  U. K. Laemmli,et al.  Cleavage of structural proteins during , 1970 .

[21]  M. Taylor,et al.  Large-scale production and purification of recombinant Galanthus nivalis agglutinin (GNA) expressed in the methylotrophic yeast Pichia pastoris , 2003, Biotechnology Letters.

[22]  S. Gaikwad,et al.  pH-dependent aggregation of oligomeric Artocarpus hirsuta lectin on thermal denaturation. , 2003, Biochemical and biophysical research communications.

[23]  P. Rougé,et al.  Structural basis for the unusual carbohydrate-binding specificity of jacalin towards galactose and mannose. , 2002, The Biochemical journal.

[24]  I. L. Ainouz,et al.  Lectins from seeds of jack fruit (Artocarpus integrifolia L.): isolation and purification of two isolectins from the albumin fraction , 1981, Biologia Plantarum.

[25]  P. Rougé,et al.  The size, shape and specificity of the sugar-binding site of the jacalin-related lectins is profoundly affected by the proteolytic cleavage of the subunits. , 2002, The Biochemical journal.

[26]  M. Hearn,et al.  Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production , 2005, Journal of molecular recognition : JMR.

[27]  P. Proost,et al.  Expression of the nucleocytoplasmic tobacco lectin in the yeast Pichia pastoris. , 2007, Protein expression and purification.