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 Smith

Research output: Contribution to journalArticle

20 Citations (Scopus)

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
StatePublished - 1996
Externally publishedYes

Fingerprint

Galactosidases
Cloning
Blood Group Antigens
Soybeans
Glycine
Organism Cloning
Complementary DNA
Erythrocytes
Molecular mass
Amino Acid Sequence
Genes
Affinity chromatography
Amino Acids
Phaseolus
Pichia
Hemagglutinins
Chromatography
Galactose
Affinity Chromatography
Population Groups

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Molecular Biology

Cite this

Cloning, expression and characterization of a blood group B active recombinant α-D-galactosidase from soybean (Glycine max). / Davis, Melissa O.; Jane Hata, D.; Johnson, Scott A.; Walker, John C.; Smith, Daniel.

In: Biochemistry and Molecular Biology International, Vol. 39, No. 3, 1996, p. 471-485.

Research output: Contribution to journalArticle

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