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To confirm that the hdcA gene from Staph. capitis IFIJ12 encodes a functional HDC， we expressed this gene in E. coli following the strategy described in the Materials and Methods section， consisting in amplifying the genes by PCR and clonin... To confirm that the hdcA gene from Staph. capitis IFIJ12 encodes a functional HDC， we expressed this gene in E. coli following the strategy described in the Materials and Methods section， consisting in amplifying the genes by PCR and cloning the products under the control of the T7 RNA polymerase-inducible /10 promoter. Cell extracts were used to detect the presence of hyperproduced proteins by SDS-PAGE analysis. Control cells containing the pT7-7 vector plasmid alone did not show expression over the 3-h time course analysed， whereas expression of additional 34Æ2-kDa protein was apparent with cells harbouring pAM28 (Fig. 2ai). In addition， cell extracts from E. coli JM109(DE3) (pLysS) cells harbouring the recombinant plasmid pAM28 were able to decarboxylate the histidine present in the reaction to histamine， whereas extracts prepared from control cells containing the vector plasmid alone did not. Figure 2(bi) shows a TLC analysis of the enzymatic reaction. Thus， we could prove experimentally that the hdcA gene encodes a functional HDC. As the protein was cloned containing a purification poli-His tag， HDC was purified on a His-TrapTM-FF crude chelating column and eluted with a stepwise gradient of imidazole. Highly purified HDC protein was obtained from pAM28 (Fig. 2aii). The eluted HDC protein was dialysed to eliminate the imidazole， and checked for HDC activity. TLC analysis demonstrated that highly purified HDC protein was able to decarboxylate histidine to form histamine (Fig. 2bii). The predicted sequence of the HDC was aligned with HDC proteins from Gram-positive bacteria (Fig. S1). As deduced from the HDC alignment， most of the residues implicated in catalysis and substrate binding in the HDC from Lactobacillus 30a (Gallagher et al. 1989) are conserved in the Staph. capitis enzyme. However， the residue Ala-260， forming the hydrophobic pocket， is not conserved in the Staph. capitis HDC protein， and a Gly residue is present in its place. In addition to the wild-type HDC enzymes， a number of mutants that produce partially active or inactive enzymes have been isolated (Recsei and Snell， 1982， Van Poelje et al. 1990). More interestingly， mutant 3 of HDC from Lactobacillus 30a， which produces a full-length protein that is slowly autoactivated， shows only one amino acid replacement at position 58 (G58A)， the Gly amino acid residue is conserved at this position in all HDC， with exception of Staph. capitis HDC with a Asn residue present in its place. An autoactivation assay was performed in order to know if S. capitis HDC follows a similar slow autoactivation. The result shows that along incubation， Staph. capitis HDC seems to be degraded instead to be autocleaved into an a chain (23 kDa) and a β chain (11.5 kDa) (Fig. 3).