A kind of preparation method of melittin
A technology of melittin and calmodulin, applied in the field of protein biosynthesis, can solve the problems of impossibility, high cost, death, etc.
Active Publication Date: 2019-01-15
北京蜜品会商贸有限公司
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], but the amount extracted by this method is very small, and it becomes almost impossible due to the limitation of the number of bees and their own weight
The second way is solid-phase synthesis, which is relatively expensive to obtain peptides
Since the N-terminal of melittin is hydrophobic, it can be inserted into the plasma membrane of the cell to kill bacteria, so melittin itself has certain toxicity and cannot be directly obtained by genetic engineering methods
So far, there is no low-cost, high-efficiency report on the preparation of melittin
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[0027] Firstly, the base sequences of calmodulin, smt3 and melittin are constructed on the vector pET-30a by genetic engineering technology, so as to obtain the recombinant plasmid. The correct result of gene sequencing proved that we successfully constructed the expression plasmid of pET-30a-calmodulin-smt3-melittin. Gene sequence translated into protein sequence see figure 1 .
[0028] Pick a single colony after transformation with Transsetta(DE3)pLysS as host bacteria, culture overnight in LB medium, inoculate into 500mL improved TB medium at a ratio of 1:100, and culture at 37°C until OD~0.6, Add IPTG at a final concentration of 0.1 mM, induce overnight at 20°C, centrifuge after 12 hours, and collect the bacteria. Resuspend the bacteria in buffer A (50mM Tris, 200mM NaCl, 5mM β-mercaptoethanol, pH 8.0) according to 5mL / g wet bacteria, add a small amount of lysozyme, DNaseI and PMSF (final concentration is 1.0mM), and stir at 4°C for half After 1 hour, the cells were ult...
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Abstract
The invention relates to the technical field of protein synthesis, in particular to a preparation method of melittin. The method comprises the following steps that 1, recombinant plasmid composed of nucleotide sequences and expression vectors of calmodulin, smt3 (SUMO family protein) and the melittin is transformed to an escherichia coli strain; 2, the strain obtained in the first step is inoculated into a TB culture medium or an improved TB culture medium, and IPTG overnight inducible expression is conducted; 3, the thallus obtained in the second step is subjected to ultrasonication and centrifugation, Ni-NTA affinity chromatography and sumo protease enzyme digestion are conducted on supernatant, the melittin with the purity reaching up to 95% is obtained through Ni-NTA affinity chromatography and Sephadex G-25 molecular sieve separation on the condition that 1.0 mM EDTA exists, and the yield can reach 16 mg melittin per liter of the melittin. According to the preparation method of the melittin, the detects that at present, the yield of the thallus is low, the cost is high, and application requirements of the melittin cannot be met can be overcome, and the method is the expressing method which is high in yield and low in cost.
Description
Technical field: [0001] The invention relates to the technical field of protein biosynthesis, in particular to a method for efficiently preparing melittin by adopting bioengineering technology. technical background: [0002] Melittin, also known as melittin, is the main active ingredient in the venom of the Italian honey bee (Apis mellificera). The small peptide consists of 26 amino acid residues, has good water solubility, has a relative molecular weight of about 2.8KDa, and does not contain disulfide bonds. Its amino acid sequence is GIGAVLKVLTTGLPALISWIKRKRQQ-NH2, and the basic amino acids are mainly concentrated at the C-terminus of the peptide (KRKR). Melittin can affect the signal transduction process of cells through a variety of ways; it has anti-tumor effects such as inducing apoptosis, antibacterial, antiviral, direct killing, immune regulation, inducing apoptosis, inhibiting metastasis and angiogenesis. In addition, the injections made of melittin have obvious e...
Claims
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IPC IPC(8): C12N15/70C07K14/435
CPCC07K14/43572
Inventor 赵亚琴杨斌盛
Owner 北京蜜品会商贸有限公司