PMID: 8828798Jun 1, 1996

Cloning, expression and characterization of a blood group B active recombinant alpha-D-galactosidase from soybean (Glycine max)

Biochemistry and Molecular Biology International
M O DavisD S Smith

Abstract

A cDNA encoding soybean alpha-D-galactosidase [E.C. 3.2.1.22] was obtained by screening a soybean library with Phaseolus alpha-D-galactosidase cDNA. The Glycine max alpha-D-galactosidase cDNA is 1.75 kb long and contains untranslated 5' and 3' sequences. The deduced amino acid sequence of the soybean gene has a high degree of homology with other eucaryotic alpha-D-galactosidases. Recombinant alpha-D-galactosidase (rGal) was expressed in Pichia pastoris and purified by affinity chromatography. Purified rGal was homogeneous as judged by SDS-PAGE analysis with the relative molecular mass under reducing conditions of 39.8, and under nonreducing conditions 38.0 kDa. The expressed protein contained the sequence NGLGHTPPMG at the N-terminus, corresponding to the deduced amino acid sequence of the soybean gene. The relative native molecular mass by Sephacryl S-200 chromatography was determined to be 33.1 kDa. The specific activity was 295.6 mumoles of PNP-alpha-D-galactopyranoside hydrolyzed per mg pure rGal per min. rGal was highly specific for alpha-D-galactosyl residues. No detectable hemagglutinin or protease activity was present in the preparations. Furthermore, rGal was active against the blood group B antigen in native human ery...Continue Reading

References

Nov 14, 1997·Current Opinion in Biotechnology·C P Hollenberg, G Gellissen
Nov 15, 2014·Critical Reviews in Biotechnology·Irina Yu BakuninaAntonio Trincone

Related Concepts

Glycine max
Blood Group H Type 1 Antigen
SDS-PAGE
Proteins, Recombinant DNA
Homologous Sequences, Amino Acid
Sequence Determinations
Substrate Specificity
Galactosides
Beano
Erythrocytes

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