The sequence of the gene for Proteus vulgaris chondroitinase ABC was reported by Ryan et al. (1994) (Entrez accession number AAB43331 = gi1828877) and confirmed by Prabhakar et al. (2005a) (link). The encoded sequence was also confirmed from the protein crystal structure by Huang et al. (2003) (link). These studies showed that an independent sequence reported by Sato et al. (1994) (link) contained several errors.
We obtained a cDNA for P. vulgaris chondroitinase ABC in a prokaryotic expression vector, although the coding region lacked an N-terminal signal sequence to direct secretion from cells, and had two inactivating mutations. Apart from these changes, the clone encoded the same sequence reported by Ryan et al. (1994) . We corrected the two mutations by site-directed mutagenesis, and added a eukaryotic signal sequence from mouse matrix metalloprotease 2 (GenBank accession no. NM008610 = gi47271505) (Reponen et al., 1992 (link)). An optimised Kozak sequence was also inserted at the 5′ end to allow recognition by eukaryotic ribosomes and to maximise protein yield. We further changed some of the codons that are unfavourable for translation by mammalian ribosomes, replacing them with those used more frequently by mammalian cells. The resulting ‘initial sequence’ is shown inSupplementary Fig. S1 .
This modified cDNA was subcloned into the eukaryotic expression vector pcDNA 3.1 (Invitrogen), in which transcription is directed by the cytomegalovirus promoter, which directs high-level expression in a wide range of mammalian cells. This initial clone is named C4.
Protein structure was visualised using RasMol Molecular Graphics Visualisation Tool (by Roger Sayle:http://www.rasmol.org ).
We obtained a cDNA for P. vulgaris chondroitinase ABC in a prokaryotic expression vector, although the coding region lacked an N-terminal signal sequence to direct secretion from cells, and had two inactivating mutations. Apart from these changes, the clone encoded the same sequence reported by Ryan et al. (1994) . We corrected the two mutations by site-directed mutagenesis, and added a eukaryotic signal sequence from mouse matrix metalloprotease 2 (GenBank accession no. NM008610 = gi47271505) (Reponen et al., 1992 (link)). An optimised Kozak sequence was also inserted at the 5′ end to allow recognition by eukaryotic ribosomes and to maximise protein yield. We further changed some of the codons that are unfavourable for translation by mammalian ribosomes, replacing them with those used more frequently by mammalian cells. The resulting ‘initial sequence’ is shown in
This modified cDNA was subcloned into the eukaryotic expression vector pcDNA 3.1 (Invitrogen), in which transcription is directed by the cytomegalovirus promoter, which directs high-level expression in a wide range of mammalian cells. This initial clone is named C4.
Protein structure was visualised using RasMol Molecular Graphics Visualisation Tool (by Roger Sayle:
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