| Objective: Our institute has screened an enzyme capable of detoxifying AFB1, named Aflatoxin-detoxifizyme (ADTZ), from the E-20 strain. The full-length E123 has been cloned and its extracellular expression in the Pichia pastoris expression system has been achieved. Research on its detoxification mechanism revealed that the ADTZ is an oxygenase that is involved in a redox reaction, but whether this uniqueness is related to its detoxification property was not depicted. X-ray crystallography and NMR are alternatives, but their high cost and long time is an obstacle. Hence, we decided to adopt the method of theoretical prediction to achieve speedy revelation of protein structure. The GFP reporter gene system has proved to be efficient in protein structure and interaction analysis in the past decade. Structural biology in combination with bioinformatics studies, protein structure prediction became a heated topic in this field abroad. Therefore, this thesis utilized the distinct characteristics of the proGFP protein folding reporter vector, and screened for ADTZ fragments with soluble domains via high-throughput screening. Analyzing results using bioinformatics tools provides information for structural characteristics of full-length ADTZ and its soluble domains.Methods: (1) Mutation of ADTZ Hindâ…¢site: we used overlap extension PCR to achieve site-specific mutation at the ADTZ Hindâ…¢site, provided that the amino acid sequence is unaltered. (2) Preparation of random ADTZ fragments: we used T-PCR (PCR amplification with tagged random primers) to obtain random fragments of the ADTZ gene, where an enzymatic cleavage site is also inserted for convenience in later experiments. (3) Screening of ADTZ soluble domains: based on the mechanism that GFP fluorescence illumination depends on its correct folding, which in turn is influenced by the correct folding of the upstream moiety, this reporter system is used to screen for ADTZ soluble domains. (4) Structural and functional analysis: we used bioinformatics tools to analyze the structural and functional properties of the ADTZ. (5) Analysis of the biological information of the ADTZ soluble domains.Results: (1) We successfully mutated the ADTZ HindIII site, providing template for later experiments. (2) We used T-PCR to amplify many ADTZ fragments. (3) By the combined technology of T-PCR and GFP, we successfully screened for soluble ADTZ fragments: ADTZ-sub 3 (residues 259-394), ADTZ-sub 5 (residues 101-194) and ADTZ-sub 6 (residues 150-193). (4) Based on results from structural informatics studies on its secondary structure, hydrophobic region(s), folding, globular region(s) and conformational changes, etc., we discovered three reliable folding regions: Region 1 (residues 1-175), Region 2 (residues 225-425) and Region 3 (residues 500-695). (5) Functional prediction reveals that ADTZ contains the same HEXXXH domain as the M49 metalloprotease family proteins, which exhibit dipeptidyl peptidaseâ…¢activity and catatyse of the release of an N-terminal dipeptide comprising four or more residues. Whether this activity is related to the detoxification of AFB1 needs to be further investigated. (6) Comparing sequences of the screened fragments and results of bioinformatics studies prevailed that the HEXXXH domain is dominant in Region 2 and Region 3. Further analysis revealed that most domains lie in Region 1 while others lie in Region 2 and 3. This information is relevant to further experiments.Conclusion: This research provides a method to investigate the structure and function of a protein with low homology. It provided much information for further research on the ADTZ. We successfully screened soluble fragments by the combined technology of T-PCR and GFP, while reducing the cost of protein purification and crystallography and complex biological analysis. This research screened out relevant soluble ADTZ fragments for further studies on its binding centre and catalytic centre.
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