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KMID : 0358920060330020304
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Journal of the Korean Academy of Pedodontics
2006 Volume.33 No. 2 p.304 ~ p.310
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MOLECULAR CLONING AND SEQUENCE ANALYSIS OF THE GENE FOR THE HEMIN-BINDING PROTEIN FROM Prevotella intermedia
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Kim Shin
Kim Sung-Jo
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Abstract
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Prevotella intermedia is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. This study has identified a hemin-binding P. intermedia protein by expression of a P. intermedia genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. The genomic library of P. intermedia was constructed into plasmid pUC18, transformed into Escherichia coli strain DH5^(α), and screened for recombinant clones using hemin-binding activity by plating onto hemin-contraining agar. Approximately 5,000 recombinant E.coli colonies were screened onto LB-amp-hemin agar, single clone(pHem1) was exhibited a clearly pigmented phenotype. The 2.5 kb insert DNA of pHem1 was determined by restriction enzyme mapping. Southern blot analysis of BamHⅠ, BgIⅡ, EcoRⅠ, HIndⅢ and PstⅠ-digested P. intermedia DNA indicated that single copy of the gene was present in the genome. Northern blot analysis revealed that the size of transcript was approximately 1.8 kb. The cloned gene contained a single ORF, consisting of approximately 850-residue amino acids. A BLAST search of the Institute for Genomic Research genes with similar nucleotide sequence revealed no significant similarity. It need further investigation to clarify the mechanisms of heme uptake in P. intermedia.
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KEYWORD
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Hemin, Hemin-binding protein, Prevotella intermedia, Gene cloning
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