Method for high-level expression of bacterial laccase in Escherichia coli
A high-efficiency expression and Escherichia coli technology, applied in the field of bioengineering, can solve the problems affecting the development and application of bacterial laccase, and achieve the effect of soluble expression and short fermentation time
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[0020] Example 1: Expression of Klebsiella soil 601 bacterial laccase MCO in E. coli
[0021] Firstly, restriction enzymes BamHI and SalI were used to digest the DNA encoding the Klebsiella soil 601 bacterial laccase gene and pMAL-c2x plasmid DNA, respectively, and the digestion buffer was 10×T reaction buffer. Digestion at 37°C for 4 hours. The digestion mixture was subjected to 1% agarose gel electrophoresis to recover a 1.6 kb DNA fragment encoding the Klebsiella soil 601 bacterial laccase gene and a 6.67 kb linear pMAL-c2x plasmid DNA. Then the two recovered DNA fragments were mixed at a ratio of 5 times the laccase gene DNA fragment:1 times the linear pMAL-c2x plasmid DNA, and used T 4 DNA ligase was ligated overnight at 16°C. Then, the ligation reaction mixture was directly transformed into E. coli DH10B, and positive transformants were screened with LB plates containing 100 μg / ml ampicillin. The size of the recombinant plasmid contained in the positive transformant was 8...
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[0022] Example 2: Expression of Klebsiella soil 601 bacterial laccase MCO mutant in E. coli
[0023] The same method was used to express the Klebsiella soil 601 bacterial laccase MCO mutant α351-378 / β351-378 in Escherichia coli DH10B. The soluble fusion protein in the supernatant was 60%, and the precipitate accounted for 40%. The DNA encoding Klebsiella soil 601 bacterial laccase gene was ligated to the ptac85 expression vector using restriction endonuclease BamHI and SalI restriction sites and expressed in E.coli Top10 bacteria. The supernatant was soluble and fused Protein is 0 and 100% in precipitation.
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[0024] Example 3: Expression of soil Hertzia 531 bacterial laccase MCO in E. coli
[0025] The restriction endonucleases EcoRI and SalI were used to digest the gene DNA and pMAL-c2x plasmid DNA encoding the H. soil 531 bacterial laccase MCO, respectively. The digestion buffer was 10×T reaction buffer. Digestion at 37°C for 4 hours. After the digestion mixture was electrophoresed on a 1% agarose gel, a 1.6 kb DNA fragment encoding the laccase gene of H. soil bacteria 531 and a 6.67 kb linear pMAL-c2x plasmid DNA were recovered. Then, the two recovered DNA fragments were mixed at a ratio of 5 laccase gene DNA fragments: 1 linear pMAL-c2x plasmid DNA, and ligated overnight at 16°C with T4 DNA ligase. Then the ligation reaction mixture was directly transformed into E. coli DH10B, and positive transformants were screened on an LB plate containing 100 μg / ml ampicillin. According to the above steps C-E to express the target protein, the soluble fusion protein in the supernatant is abo...
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