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) c ells has been cloned and expressed. Completely degenerate oligonucleot ide primers, which were based on the amino acid sequence of peptides o btained from the purified sialidase (Warner et al., Glycobiology, 3, 4 55-463, 1993), and the polymerase chain reaction, with single-stranded cDNA template, were employed to generate a unique oligonucleotide pro be. The unique probe of 93 bp was used for screening a lambda gt 10 CH O 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 isolate d 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 regio n. No signal peptide was identified in the insert, suggesting a cytoso lic localization for the protein. No significant primary sequence iden tities were observed when the deduced amino acid sequence of the CHO c ell sialidase was compared with other mammalian proteins or microbial sialidases. However, the protein had significant sequence alignment si milarity with several bacterial sialidases. Two 'Asp box' motifs in th e CHO cell sialidase had a remarkable alignment positioning in the pro tein sequence with the similar motifs of the Salmonella LT2 and Clostr idium perfringens sialidases. High levels of the enzyme were expressed in Spodoptera frugiperda cells infected with a modified Autographa ca lifornica nuclear polyhedrosis virus harbouring the sialidase cDNA.