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Establishment Of Cell Surface Display System Based On INP N-domain Of Xanthomonas And Its Preliminary Application

Posted on:2010-07-15Degree:MasterType:Thesis
Country:ChinaCandidate:W FuFull Text:PDF
GTID:2120360302455310Subject:Microbiology
Abstract/Summary:PDF Full Text Request
The N-domain(541bp) of ice nucleation protein gene from Xanthomonas campestris pv. campestris ACCC 10049, inaXN, was cloned in this work, and it's showed 99% homology to that of X. campestris ATCC 33913 via the alignment with NCBI database. Meanwhile, it was anticipated that inaXN could be used as an anchoring motif for bacterial surface display, which based on the analysis and prediction to its physico-chemical properties, secondary structure and transmembrane domains.In order to determine whether the inaXN can act as anchoring motif for cell surface display or not, a recombinant plasmid pETinaXN-bpL was constructed in the way that bpL lipase from Bacillus pumilus YZ02, as a passenger protein, was fused into inaXN and cloned to plasmid pET28a(+). The recombinant plasmid pETinaXN-bpL was then transformed into Escherichia coli Rosetta 2(DE3) and recombinant strains PIL3R was obtained. The localization of fusion protein of inaXN-bpL on the bacterial cell surface was confirmed by cell fractionation, lipase activity assay, InvisionTM His-tag In-gel stain analysis and immunofluorescence microscopy. Lipase activity was assayed by a spectrophotometric method, using p-nitrophenyl palmitate as substrate. It was found that the activity of displayed lipase was as high as 74.6±4.5 U/mg lyophilized cell, the number of lipase molecules displayed per cell was estimated up to 19267. Importantly, the optimal pH increased from 8.5 to 11.0 compared with the free lipase, and the displayed lipase still retained over 80% of full activity after incubation at 40℃in Tris-HCl buffer for a week.Lastly,α-domain tetrameric polymer of monkey metallothionein (MT4α), showing strong ability to absorb heavy metal, was chosen to act as a passenger protein. Fusion gene of inaXN-MT4αwas inserted into plasmid pTPG-PnifH and its expression was controlled by a symbiotic nitrogen fixation promoter PnifH. The construction of recombinant plasmid pIM-PnifH build up the basis for the construction of engineered rhizobia to be used for the bioremediation of heavy metal contamination in soil.
Keywords/Search Tags:surface display, Xanthomonas campestris, lipase, whole-cell biocatalysis, ice nucleation protein, metallothionein, heavy metal adsorption
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