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

Protein Expr Purif. 2003 Dec;32(2):309-16. doi: 10.1016/j.pep.2003.08.007.

Abstract

Clostridium perfringens alpha-N-acetylgalactosaminidase (alphaNAG) hydrolyzed the terminal N-acetyl-alpha-d-galactosamine from the blood type A(2) 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 alphaNAG 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 71.96kDa and the native molecular weight of 72.42kDa. 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 A(2) epitope. These characteristics were similar to those of native alphaNAG from C. perfringens. With adequate expression in E. coli, sufficient recombinant alphaNAG 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.

Publication types

  • Comparative Study

MeSH terms

  • ABO Blood-Group System / immunology
  • Antibodies, Monoclonal / immunology
  • Antibodies, Monoclonal / metabolism
  • Chromatography, High Pressure Liquid / methods
  • Chromatography, Ion Exchange
  • Clostridium perfringens / enzymology*
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Stability
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Hexosaminidases / genetics
  • Hexosaminidases / isolation & purification*
  • Hexosaminidases / metabolism
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Osmolar Concentration
  • Recombinant Proteins / isolation & purification*
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Temperature
  • alpha-N-Acetylgalactosaminidase

Substances

  • ABO Blood-Group System
  • Antibodies, Monoclonal
  • Recombinant Proteins
  • Hexosaminidases
  • NAGA protein, human
  • alpha-N-Acetylgalactosaminidase