A kind of plectasin and its gene and preparation method
A technology of mycelia and gene, applied in the field of bioengineering, can solve the problems of low yield of mycelia, complicated and difficult purification steps, etc.
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Embodiment 1
[0055] The design of embodiment 1.pletasin gene, the construction of synthesis and expression vector
[0056] 1.1 Design and synthesis of plectasin plectasin protein gene
[0057] The inventors designed the expression vector pYG330 of the plectasin plectasin protein, which can not only enable the plectasin plectasin fusion protein to be expressed in large quantities, but also does not affect the growth of the host Escherichia coli, and is convenient for subsequent separation and purification steps. The construction method of vector pYG330 comprises the following steps:
[0058] According to the published nucleotide sequence of plectasin in Saprophytic Ascomycetes (PerH.Mygind, RikkeL.Fischer, KirkM.Schnorretal, Plectasinisapeptideantibioticwiththerapeuticpotentialfromasaprophyticfungus[J]. Nature, 437(13), October2005: 975-980), and then refer to the large intestine Bacteria codon preference usage table, designed and synthesized the gene sequence of plectasin fusion protein e...
Embodiment 2
[0066] Example 2. Induced expression of fusion protein
[0067] 2.1 Plectasin fusion protein
[0068] The plectasin fusion protein of the present invention has a molecular weight of 25KD, which is co-expressed by a part of the protein expressed by the universal expression vector itself and the fragment inserted by the inventor, and terminates at the terminator TAG designed by the inventor, and the 4.7KD target protein is located at The end of the fusion protein, which is a part of the fusion protein as a whole, can be cut at the designed TEV restriction site to obtain the 4.7KD target protein. Among them, the 20.3KD large peptide is encoded by the nucleotide sequence of the expression vector backbone plasmid, and also contains the His-Tag tag designed by the inventor for easy separation and purification, and the amino acid sequence of the TEV enzyme recognition site.
[0069] 2.2 Induced expression of plectasin fusion protein
[0070] 1) Optimization of the concentration of ...
Embodiment 3
[0076] Example 3. Fusion protein purification
[0077] According to the optimized induction conditions, the transformed cells obtained in Example 1 were induced and cultivated in 250mL LB medium to express the fusion protein of interest. 10000rpm, 4°C, 10min centrifugation to collect the bacterial cells; wash once with PBS, add 10ml of binding buffer NTA0, ice bath, ultrasonic cracking (500W, 5s / 5s), 10000rpm centrifugation for 25min, take the supernatant, filter with a 20μm filter membrane, Automatic loading to AKTA affinity chromatography column,
[0078] bufferA is 20mMNa 3 PO 4 , 500mMNaCl, 40mM imidazole, pH=7.4;
[0079] bufferB is 20mMNa 3 PO 4 , 500mM NaCl, 500mM imidazole, pH=7.4.
[0080] Carry out gradient elution with the eluent (60mM, 80mM, 100mM, 200mM) containing the following concentrations of imidazole in turn, collect the eluent for electrophoresis detection, use the gel protein analysis software (Quantityone4.6.2) to calculate the purity, and determine t...
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