Expression of frutalin, an α-d-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris

Abstract

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.

Crystallization and preliminary X-ray diffraction studies of frutalin, an α-D-galactose-specific lectin fromArtocarpus incisaseeds

“…The SDS-PAGE showed a typical jacalin-like lectin mass pattern with two bands between 20 and 14 kDa (Fig. 1), which correspond to a glycosylated fraction and a slightly or nonglycosylated fraction, respectively (Oliveira et al, 2008). The deconvoluted FTL mass spectrum showed different masses at approximately 16.5 kDa, which is consistent with the presence of isoforms of identical monomers.…”

“…3). We used the coordinates of the jacalin monomer, which shares 91% sequence identity with FTL, as a search model (PDB entry 3p8s; Oliveira et al, 2008;Oliveira, Costa et al, 2009). The best solution was obtained in space group I2 with four monomers per asymmetric unit, which yielded an R factor, LLG and TFZ of 38.6%, 11 514.0 and 19.9, respectively.…”

Modification of paper properties using carbohydrate-binding module 3 from the Clostridium thermocellum CipA scaffolding protein produced in Pichia pastoris: elucidation of the glycosylation effect

“…Transformants were selected on YPD-sorbitol plates (2 % glucose, 2 % peptone, 1 % yeast extract and 1 M sorbitol) containing 100 lg/ml of Zeocin and incubated at 30°C for 3 days. Insertions were confirmed by ''colony PCR'', as described elsewhere (Oliveira et al 2008). Positive PCR Cellulose transformants were inoculated on YPD plates containing 1000 lg/ml of Zeocin, for the selection of multicopy integrated strains.…”

cDNA Cloning and Functional Expression of the α-d-Galactose-Binding Lectin Frutalin in Escherichia coli

“…Cells were disrupted by sonication on ice (Branson Sonifier, 5 min, duty cycle 50%, output 5) and the insoluble debris recovered by centrifugation (30 min, 10,000g, 4°C). The E. coli soluble and insoluble fractions were analysed for the presence of recombinant frutalin by denaturing SDS-PAGE electrophoresis and Western blot analyses, as previously described [10]. The primary antibody was polyclonal anti-frutalin, the secondary antibody was ''anti-rabbit IgG peroxidase'' (Sigma) and DAB (3,3 0 -diaminobenzidine, Sigma) was used to specifically detect membrane-bound secondary antibody.…”

“…The isoforms of frutalin, having different binding affinities and/or specificities, may introduce additional unwanted variability when used, for instance, as a diagnostic tool. The methylotrophic yeast Pichia pastoris has already been successfully used for frutalin production, using an optimised synthetic gene encoding a mature frutalin protein sequence [10]. The bacteria Escherichia coli was also chosen as a host for frutalin production as this expression system offers a rapid and economical way to produce unglycosylated recombinant frutalin.…”