Biological fermentation preparation method for active peptide with skin whitening effect
An active polypeptide and bio-fermentation technology, which is applied in the field of bio-fermentation preparation of active polypeptides, can solve the problems of high production cost, difficult separation and purification, and many enzymatic hydrolysis products by enzymatic hydrolysis, and achieve the problem of purification cost and good biocompatibility , the effect of simple operation
Inactive Publication Date: 2015-12-23
HUAQIAO UNIVERSITY
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Problems solved by technology
The enzymatic hydrolysis method has the advantages of strong specificity, high efficiency, mild conditions, and product safety. However, due to the large number of enzymatic hydrolysis products, it is difficult to separate and purify. In addition, the enzyme itself is relatively expensive, so the production cost of the enzymatic hydrolysis method is extremely high.
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Embodiment 1
[0030] (1) Construction of fusion protein: the fusion protein includes the ELP [KV 8 F-40], linking peptide Linker, enterokinase enzyme cleavage recognition site EK and active polypeptide decapeptide-12 (P4), wherein ELP[KV 8 F-40] comprising the amino acid sequence shown in SEQID01 (its nucleotide sequence is shown in SEQID05), the connecting peptide Linker comprises the amino acid sequence shown in SEQID02, and the enterokinase restriction recognition site EK comprises the amino acid sequence shown in SEQID03 Amino acid sequence, the active polypeptide decapeptide-12 (P4) includes the amino acid sequence shown in SEQID04, and the expression vector is constructed as figure 1 and figure 2Shown: First, according to the codon bias of E. coli, the Linker-EK-P4 gene sequence was synthesized and expressed, and the Linker-EK-P4 gene sequence was spliced into pET-22b-ELP[KV 8 F-40] (the sequence shown in SEQID05 was connected to pET-22b (+) in advance), the expression vector pET...
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The invention discloses a biological fermentation preparation method for active peptide with the skin whitening effect. The method comprises the following steps that 1, fusion protein is constructed, wherein the fusion protein comprises ELP[KV8F-40], linker peptide, an enterokinase digestion recognition site and the active peptide decapeptide-12 which are connected in sequence; 2, amino acid sequences of the fusion protein are used for synthetizing gene sequences expressing the fusion protein according to the preference of codons of escherichia coli; 3, the gene sequences are spliced to an escherichia coli expression vector, the escherichia coli is guided in for induction expression, and thaluses are disrupted and centrifuging is performed to obtain a supernate; 4, on the conditions of a certain salt ion concentration and a buffer solution, the fusion protein is made to achieve phase transformation by controlling the temperature of the surpernate obtained in the third step for gathering, centrifugal purification is performed to obtain the fuse protein, and the active peptide with the skin whitening effect is obtained. The method is efficient, safe and low in cost.
Description
technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a biological fermentation preparation method of an active polypeptide with whitening effect. Background technique [0002] Decapeptide-12 (P4) is derived from basic fibroblast growth factor (Heparin-binding growth factor-2, HBGF-2), which is the 106th to 115th amino acid residue of HBGF-2, and its amino acid sequence is YRSRKYSSWY. In the study of Ubeid et al., it was found that P4 has a significant inhibitory effect on tyrosinase, and its half-inhibitory concentration IC 50 Only 50 μM, compared with 680 μM of hydroquinone, the effect is abnormally obvious, and at the same time, the cytotoxicity is extremely low (AbuUbeidA, ZhaoL, WangY, et al. Short-sequence oligopeptides with inhibitory activity against mushroom and humantyrosinase [J]. 2249). The inhibition of P4 on tyrosinase belongs to competitive inhibition, which tends to inhibit the reaction of tyros...
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IPC IPC(8): C12P21/02C12N15/70C12N15/62
Inventor 张光亚葛慧华林源清
Owner HUAQIAO UNIVERSITY