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.
Oliveira
Carla
C
IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Felix
Wagner
W
Moreira
Renato A
RA
Teixeira
José A
JA
Domingues
Lucília
L
D001483:Base Sequence; D015153:Blotting, Western; D050260:Carbohydrate Metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006386:Hemagglutination Tests; D010843:Pichia / Q000235:genetics; D037121:Plant Lectins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014170:Transformation, Genetic
D017931:DNA Primers; D037121:Plant Lectins; D011994:Recombinant Proteins; C043696:jacalin