Cloning and expression of the pneumococcal neuraminidase gene in Escherichia coli.
A gene bank of Sau3AI-generated Streptococcus pneumoniae DNA fragments was constructed in Escherichia coli K-12 by cloning into the BamHI site of the cosmid vector pHC79. One clone capable of cleaving the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetyl-neuraminic acid was isolated. This activity was inhibited by treatment with a mouse antiserum raised against purified pneumococcal neuraminidase. The recombinant plasmid purified from this clone (designated pJCP301) contained approximately 3.0 kb of pneumococcal DNA. Western-blot analysis indicated that E. coli K-12[pJCP301] produced a 98-kDa polypeptide which reacted with antineuraminidase serum.
Berry
A M
AM
Department of Microbiology, Adelaide Children's Hospital, North Adelaide, South Australia.
Paton
J C
JC
Glare
E M
EM
Hansman
D
D
Catcheside
D E
DE
D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D009439:Neuraminidase / Q000235:genetics*; D010455:Peptides / Q000032:analysis; D010957:Plasmids; D015183:Restriction Mapping; D013296:Streptococcus pneumoniae / Q000201:enzymology / Q000235:genetics*; D014169:Transformation, Bacterial
D010455:Peptides; D004247:DNA; D009439:Neuraminidase