Cloning and expression of a soluble sialidase from Chinese hamster ovary cells: sequence alignment similarities to bacterial sialidases.
A cDNA encoding a soluble sialidase from Chinese hamster ovary (CHO) cells has been cloned and expressed. Completely degenerate oligonucleotide primers, which were based on the amino acid sequence of peptides obtained from the purified sialidase (Warner et al., Glycobiology, 3, 455-463, 1993), and the polymerase chain reaction, with single-stranded cDNA template, were employed to generate a unique oligonucleotide probe. The unique probe of 93 bp was used for screening a lambda gt 10 CHO cell cDNA library. A single clone, which contained a 1.4 kb insert, was isolated after screening 450,000 recombinants. The complete coding region of the protein, 1137 nucleotides, was contained in the isolated clone and it predicted a protein of 379 amino acids. The insert had a 186 bp 5' non-coding leader sequence and a 40 bp 3' non-coding region. No signal peptide was identified in the insert, suggesting a cytosolic localization for the protein. No significant primary sequence identities were observed when the deduced amino acid sequence of the CHO cell sialidase was compared with other mammalian proteins or microbial sialidases. However, the protein had significant sequence alignment similarity with several bacterial sialidases. Two 'Asp box' motifs in the CHO cell sialidase had a remarkable alignment positioning in the protein sequence with the similar motifs of the Salmonella LT2 and Clostridium perfringens sialidases. High levels of the enzyme were expressed in Spodoptera frugiperda cells infected with a modified Autographa californica nuclear polyhedrosis virus harbouring the sialidase cDNA.
Ferrari
J
J
Genentech, Inc., Medicinal and Analytical Chemistry Department, S. San Francisco, CA 94080.
Harris
R
R
Warner
T G
TG
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells / Q000201:enzymology*; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006031:Glycosylation; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D017924:Nucleopolyhedroviruses / Q000235:genetics; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D012995:Solubility; D018411:Spodoptera / Q000378:metabolism
D018076:DNA, Complementary; D009439:Neuraminidase