| Summary: | 碩士 === 國立中興大學 === 生物化學研究所 === 88 === XpsD is an outer membrane protein that is required for the secretion of extracellular protein in Xanthomonas campestris pv. campestris. Previous studies indicated that the XpsD protein forms a multimer, and it appears to interact with the secreted a-amylase. In this study, we conducted co-immune precipitation followed by immunoblot analysis to characterize the interaction between the XpsD protein and a-amylase. The N-domain truncated XpsDD29-428 (pCD105) protein was not precipitated by antibody against a-amylase, whereas addition of amino acid residues 29-73 and 304-428 (XpsDD74-303, pHM4) was sufficient to observe co-immune precipitation between the two proteins. These results suggested that interaction between the XpsD protein and a-amylase is probably through the N-domain of the XpsD. Subsequent attempt to investigate the significance in the region missing in XpsDD74-303 revealed a short region within amino acid residues 218-224 that was shared by two truncated XpsD proteins and exhibited enhanced co-immune precipitation. To investigate the possible involvement of this region in specific recognition between the XpsD and a-amylase, I constructed three 3-amino acid deletions and two insertions in the full-length XpsD and the C-domain truncated XpsDD448-650 and conducted further co-immune precipitation analysis. The results indicated that co-immune precipitation between the full-length XpsD and the C-domain truncated XpsDD448-650 with a-amylase are probably through different modes of interaction. Furthermore, deletions within amino acid residues 218-224 did not cause significant effect upon co-immune precipitation between the full-length XpsD and a-amylase. Reduction of co-immune precipitation between the full-length XpsD and a-amylase caused by insertion of the myc epitope downstream of the amino acid residue 222 was not great enough to suggest a specific recognition region located within this region. None of the results from this study is supportive of a specific recognition region in the N-domain of the XpsD protein in the interaction between XpsD and a-amylase. Perhaps the N-domain of the XpsD protein is required for maintaining the correct overall structure to allow passage of a-amylase through the channel.
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