Identification, molecular cloning and expression of an alpha-N-acetylgalactosaminidase gene from Clostridium perfringens.
The Clostridium perfringens gene encoding the previously characterized alpha-N-acetylgalactosaminidase (alphaNAG) was identified by protein microsequencing and database searching. The alphaNAG protein, designated AagA, was found to be encoded by a hypothetical gene of unknown function in the recently completed genome sequence of C. perfringens strain 13. The deduced translation product of 629 amino acid residues possessed a region of limited homology to several hypothetical open reading frames, an enterotoxin of unknown function and several known or predicted alpha-galactosidases, but did not exhibit homology to any of the multiple sequenced eukaryotic alphaNAG proteins. The C. perfringens aagA gene, encoding AagA, was cloned in an Escherichia coli T7 expression system, resulting in recombinants exhibiting high-level expression of the expected alphaNAG activity. To our knowledge, this is the first report of the cloning and expression of a bacterial alphaNAG-encoding gene and represents an important step in the development of recombinant alphaNAG as a tool in the enzymatic conversion of blood group antigens.
Department of Veterinary Pathobiology, University of Missouri-Columbia, 65212, USA.
Michael J
MJ
Calcutt
Hsin-Yeh
HY
Hsieh
Linda F
LF
Chapman
Daniel S
DS
Smith
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006596:Hexosaminidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D048809:alpha-N-Acetylgalactosaminidase
D011994:Recombinant Proteins; D006596:Hexosaminidases; D048809:alpha-N-Acetylgalactosaminidase