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

Melissa O. Davis, D. Jane Hata, Scott A. Johnson, John C. Walker, Daniel S. Smith

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

A cDNA encoding soybean α-D-galactosidase [E.C. 3.2.1.22] was obtained by screening a soybean library with Phaseolus α-D-galactosidase cDNA. The Glycine max α-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 α-D-galactosidases. Recombinant α-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 μmoles of PNP-α-D-galactopyranoside hydrolyzed per mg pure rGal per min. rGal was highly specific for α-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 erythrocyte cell suspension assays. The only detectable erythrocyte phenotypic change was loss of the B and P1 epitopes. Consequently, recombinant Glycine max α-D-galactosidase may have useful biotechnical applications in the potential mass production of universally transfusable type O erythrocytes by enzymatic conversion.

Original languageEnglish (US)
Pages (from-to)471-485
Number of pages15
JournalBiochemistry and Molecular Biology International
Volume39
Issue number3
DOIs
StatePublished - Jan 1 1996

Keywords

  • Recombinant α-D-galactosidase
  • Type O red blood cells

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Genetics

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