Cloning, expression, and characterization of the Micromonospora viridifaciens neuraminidase gene in Streptomyces lividans.
We have cloned the Micromonospora viridifaciens neuraminidase (EC 3.2.1.18) gene (nedA) in Streptomyces lividans. This was accomplished by using the vector pIJ702 and BglII-BclI libraries of M. viridifaciens chromosomal inserts created in S. lividans. The libraries were screened for the expression of neuraminidase by monitoring the cleavage of the fluorogenic neuraminidase substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetyl-neuraminic acid. Positive clones (BG6, BG7, BC4, and BC8) contained the identical 2-kb BclI-BglII fragment and expressed neuraminidase efficiently and constitutively using its own promoter in the heterologous host. From the nucleotide sequence analysis, an open reading frame of 1,941 bp which encodes a polypeptide with an M(r) of 68,840 was detected. The deduced amino acid sequence has five Asp boxes, -Ser-X-Asp-X-Gly-X-Thr-Trp, showing great similarity to other bacterial and viral neuraminidases. We have also identified the catalytic domain by using truncated proteins produced in S. lividans.
Sakurada
K
K
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Japan.
Ohta
T
T
Hasegawa
M
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D015723:Gene Library; D005798:Genes, Bacterial / Q000235:genetics*; D005822:Genetic Vectors; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D008847:Micromonospora / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000235:genetics* / Q000302:isolation & purification; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012097:Repressor Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000235:genetics / Q000254:growth & development; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D006923:Hymecromone; C021224:2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid; D009439:Neuraminidase