Cloning, expression and characterization of a blood group B active recombinant alpha-D-galactosidase from soybean (Glycine max).
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 erythrocyte cell suspension assays. The only detectable erythrocyte phenotypic change was loss of the B and P1 epitopes. Consequently, recombinant Glycine max alpha-D-galactosidase may have useful biotechnical applications in the potential mass production of universally transfusable type O erythrocytes by enzymatic conversion.
Davis
M O
MO
Division of Biological Sciences, University of Missouri, Columbia 65212, USA.
Hata
D J
DJ
Johnson
S A
SA
Walker
J C
JC
Smith
D S
DS
D000017:ABO Blood-Group System / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000737:chemistry; D005697:Galactosides / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009994:Osmolar Concentration; D010843:Pichia / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000201:enzymology; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000737:chemistry* / Q000235:genetics
D000017:ABO Blood-Group System; D005697:Galactosides; D010940:Plant Proteins; D011994:Recombinant Proteins; D000519:alpha-Galactosidase