High-level soluble expression method and application of recombined tyrosine decarboxylase

A technology of tyrosine decarboxylase and recombinant plasmid, which is applied in the field of enzyme engineering, can solve the problems of a large number of inclusion bodies, and achieve the effects of high expression level, high recovery rate and low cost

Active Publication Date: 2013-12-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] 2. Solve the problem of a large number of inclusion bodies when rTDC is expressed by heterologous recombination in E. coli, and realize its high-level soluble expression

Method used

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  • High-level soluble expression method and application of recombined tyrosine decarboxylase
  • High-level soluble expression method and application of recombined tyrosine decarboxylase
  • High-level soluble expression method and application of recombined tyrosine decarboxylase

Examples

Experimental program
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Effect test

Embodiment 1

[0079] Example 1: Cloning and preliminary expression of Lactobacillus breve tyrosine decarboxylase TDC gene

[0080] The TDC gene used in the present invention is derived from Lactobacillus brevis Lactobacillus brevis CGMCC 1.2028, the primer design refers to the gene sequence encoding TDC in GenBank (EU195891.1), the gene length is 1881 bp, encoding 626 amino acids. The upstream primer is shown in SEQ ID No. 2, adding Nhe I enzyme cutting site; Downstream primer is shown in SEQ ID No. 3, adds in 5 ' end xho I restriction site. The TDC gene fragment was obtained by PCR amplification, and the reaction system was as follows:

[0081]

[0082] PCR reaction program: denaturation at 94°C for 10 min, denaturation at 94°C for 1 min, annealing at 57°C for 45 s, extension at 72°C for 2 min, after 30 cycles of reaction, extension at 72°C for 10 min, and finally cooling to 4°C. After PCR, 5 μL of the product was used for agarose gel electrophoresis, the agarose concentration...

Embodiment 2

[0105] Embodiment 2: the optimization of the fermentation medium of TDC recombinant bacterium and fermentation enzyme production condition

[0106] The TDC recombinant bacteria were cultured in a 250 mL shake flask containing 30 mL LB / Kan medium for 12 h, and inoculated into LBG / Kan medium at an inoculum size of 2% to induce rTDC expression. Figure 4 The expression of rTDC within 6 h of induction of the recombinant bacteria. It can be seen that with the prolongation of the induction time, both the biomass and the yield of rTDC showed an increasing trend, and the expression of protein was greatly increased compared with that before optimization. Figure 4 The fermentation conditions are: OD after transfer 600 The induction started when it reached 0.5, the IPTG concentration was 0.4 mM, the induction temperature was 25°C, and 180 rpm.

[0107] After optimizing the fermentation medium, there was almost no inclusion body formation within the first 6 hours of rTDC induction. Af...

Embodiment 3

[0115] Example 3: Purification and enzymatic property determination of recombinant tyrosine decarboxylase

[0116] All purification operations were performed at or near 4°C at low temperature.

[0117] The induced recombinant bacterial fermentation broth was centrifuged at 8000 rpm for 10 min to collect the bacterial cells. The pellet was washed twice with buffer A and then resuspended. The cells were disrupted by ultrasonication or high-pressure homogenization, and centrifuged again. The obtained supernatant was rTDC crude enzyme solution.

[0118] First equilibrate the Ni column with buffer A at a flow rate of 2 mL / min for 10 min, then load the sample at a flow rate of 2 mL / min, continue to wash with buffer A to the baseline after loading the sample, and then replace buffer B to elute rTDC , the eluent flow rate is 2 mL / min, the elution process is as follows Figure 5 shown. The purity of rTDC after purification by Ni column and the size of rTDC single subunit are as foll...

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Abstract

The invention relates to a high-level soluble expression method and application of recombined tyrosine decarboxylase, belongs to the technical field of enzyme engineering, and relates genetically engineered strain E. coliBL21(DE3) / pET24-TDC for recombined expressed lactobacillus brevis tyrosine decarboxylase, a construction method of the genetically engineered strain, a high-level soluble expression method for the TDC and application of the TDC. A TDC encoding gene is connected to an expression vector and passed to an escherichia coli expression host; glucose is added to fermentation medium; accordingly, soluble expression of the recombined TDC (rTDC) can be increased significantly, protein yield is up to 224mg / L from fermentation broth. The rTDC with His-Tag at the Ni column purified C-end is used, so that the recovery rate is 90%. The rTDC has 133.5U / mg specific enzyme activity against substrate L-tyrosine. The soluble expression method of the rTDC has the advantages of high expression level, purification simplicity, high recovery rate, low cost and the like and can be applied to the preparation of tyramine and dopamine and the treatment study on Parkinson's disease.

Description

technical field [0001] A method for soluble high expression of recombinant tyrosine decarboxylase and the application of the enzyme belong to the technical field of enzyme engineering. The invention specifically relates to the cloning of Lactobacillus brevis tyrosine decarboxylase (TDC) gene, the construction of its genetically engineered bacteria, the high-level soluble expression method of recombinant TDC and the application of the enzyme. Background technique [0002] Tyrosine decarboxylase (TDC) is a pyridoxal phosphate (PLP)-dependent decarboxylase that catalyzes the decarboxylation of tyrosine to tyramine. TDC is ubiquitous in microorganisms and plants. According to current research, the specific enzyme activity of TDC derived from microorganisms is several orders of magnitude higher than that of TDC derived from plants. At the same time, some TDCs have dopa decarboxylase (DDC) activity, which can decarboxylate dopa to generate dopamine, so the research of TDC has ref...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/60C12N15/70C12N9/88C12P13/00A61K38/51A61P25/16C12R1/19
Inventor 倪晔章凯
Owner JIANGNAN UNIV
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