Molecular cloning of the polyphenol oxidase from Escherichia coli and its heterologous high-efficiency expression
Sinapine or gossypol(phenolic compounds)as anti-nutritional factor in non-grain feed resource rapeseed meal or cottonseed meal has seriously affected their application value in animal husbandry.A strain of Escherichia coli SDB2 screened by our research group has been proved to degrade sinapine and gossyrol by secreting polyphenol oxidase.This experiment aimed to clone the SDB2 polyphenol oxidase gene and achieve its high-efficiency expression by using genetic engineering technology,laying a foundation for the large-scale application of SDB2 polyphenol oxidase in feed industry.The polyphenol oxidase gene was cloned from Escherichia coli SDB2,which was connected with plasmid pET28a and transformed into Escherichia coli BL21 receptive cells for culture.The recombinant strain was verified by PCR and plasmid digestion,and the recombinant cloned gene was analyzed by bioinformatics.A"2 ×2"cross experiment design was used to induce the high-efficiency expression of SDB2 polyphenol oxidase gene in the recombinant strain by temperature and IPTG concentration.After SDS-PAGE and WB double verification,the optimal induction conditions were established,and the target enzyme protein was finally purified.The results showed that:(1)The polyphenol oxidase gene of SDB2 was cloned successfully and the recombinant strain was constructed.(2)The recombinant cloned SDB2 polyphenol oxidase gene contained 741 target nucleotides,263 encoded amino acids(including 20 tagged amino acids),the relative molecular weight of the protein composed of amino acids was 28499.0,and the theoretical isoelectric point was 6.94.Therefore,the three-dimensional structure model of SDB2 recombinant polyphenol oxidase was predicted.(3)The optimal induction conditions for heterogenic expression of SDB2 polyphenol oxidase were 37 ℃ and 1 mmol IPTG.Under these conditions,a large number of soluble enzyme proteins were expressed,which was conducive to industrial production.The results indicated that the molecular cloning and heterologous expression of Escherichia coli SDB2 polyphenol oxidase were successfully achieved under the experimental conditions,which provided a technical reference for the efficient utilization of non-grain feed resources.
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