Purification and characterization of a recombinant α-N- acetylgalactosaminidase from Clostridium perfringens

Hsin Yeh Hsieh, Michael J. Calcutt, Linda F. Chapman, Moonmoon Mitra, Daniel Smith

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Clostridium perfringens α-N-acetylgalactosaminidase (αNAG) hydrolyzed the terminal N-acetyl-α-D-galactosamine from the blood type A2 antigen producing H antigen, blood type O. Blood type O is universally compatible in the ABO system. Purification of the native enzyme is difficult with very low yields. To obtain the enzyme in satisfactory yield, the gene encoding the clostridial enzyme was cloned in an Escherichia coli T7 expression system. A highly purified preparation of recombinant αNAG was obtained from cell lysates by ion-exchange chromatography and high-pressure liquid chromatography. The final preparation was homogeneous by SDS-PAGE with a molecular mass of 71.96 kDa and the native molecular weight of 72.42 kDa. The enzyme was highly selective for terminal N-acetylgalactosamine residues. No other significant exoglycosidase activities, particularly neuraminidase, were detected. The pH optimum of the enzyme was between 6.5 and 7.0 and activity was relatively unaffected by ionic strength. ELISA experiments demonstrated activity against blood type A2 epitope. These characteristics were similar to those of native αNAG from C. perfringens. With adequate expression in E. coli, sufficient recombinant αNAG enzyme mass can be obtained for potential use in enzymatic conversion of human blood type A 2 red blood cells to universally transfusable type O red blood cells.

Original languageEnglish (US)
Pages (from-to)309-316
Number of pages8
JournalProtein Expression and Purification
Volume32
Issue number2
DOIs
StatePublished - Dec 2003
Externally publishedYes

Fingerprint

Clostridium
Clostridium perfringens
Purification
Blood
Enzymes
varespladib methyl
Acetylgalactosamine
Escherichia coli
Erythrocytes
Cells
High pressure liquid chromatography
Glycoside Hydrolases
Ion Exchange Chromatography
Neuraminidase
Gene encoding
Osmolar Concentration
Molecular mass
Chromatography
Ionic strength
Epitopes

Keywords

  • α-N-acetylgalactosaminidase
  • ABO blood group
  • Blood type A epitope
  • Clostridium perfringens
  • Seroconversion

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and characterization of a recombinant α-N- acetylgalactosaminidase from Clostridium perfringens. / Hsieh, Hsin Yeh; Calcutt, Michael J.; Chapman, Linda F.; Mitra, Moonmoon; Smith, Daniel.

In: Protein Expression and Purification, Vol. 32, No. 2, 12.2003, p. 309-316.

Research output: Contribution to journalArticle

Hsieh, Hsin Yeh ; Calcutt, Michael J. ; Chapman, Linda F. ; Mitra, Moonmoon ; Smith, Daniel. / Purification and characterization of a recombinant α-N- acetylgalactosaminidase from Clostridium perfringens. In: Protein Expression and Purification. 2003 ; Vol. 32, No. 2. pp. 309-316.
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