Preparation method of multi-copy golden pomfret flavor peptide, expression vector and recombinant bacteria

Abstract

The invention discloses a preparation method of multi-copy golden pomfret flavor peptide. The preparation method is characterized by comprising the following steps of: 1) designing and synthesizing amulti-copy tandem flavor peptide genome and amplifying; 2) connecting the amplified flavor peptide genome to a plasmid with a His-sumo label to obtain a recombinant expression vector; 3) transformingthe expression vector into host bacteria, screening and then inducing expression; 4) performing preliminary purification to obtain flavor peptide with the His-sumo label; and 5) cracking the flavor peptide with the His-sumo label to remove the His-sumo label, and performing secondary purification to obtain the flavor peptide. The invention also provides the expression vector containing the flavorpeptide genome, recombinant bacteria and application thereof. The flavor peptide prepared according to the invention is quick, simple and convenient, has high cloning efficiency and can quickly and effectively remove the influence of a label sequence on the flavor peptide.

Description

Technical field:[0001]The present invention relates to the field of food additives, and more particularly to a preparation method of multi-copy golden fresh taste peptide and expression vectors and recombinant bacteria.Background technique:[0002]The savory is one of the five basic tastes, which can make people a pleasant taste experience. Tasty peptide is a new peptide flavor after the sodium oxamine, gobinic acid and the vibrates, etc., not only with delicious tastes, but also provides amino acid nutrients, and fresh tile peptides in condiments. The field has huge market potential.[0003]Tasty peptide large-scale production is the basis for its wide application in the field of condiments. In recent years, the use of recombinant cloning expressions have been gradually developed. Most of the microbial expression of fresh tamper peptides is based on BMP. The relevant reports of other taste peptides are very limited, and this area is still in the starting stage. This technique mainly cl...

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Problems solved by technology

[0004] Wang Yanping and others used the secretory expression plasmid pPIC9 as a vector to integrate multiple copies of the BMP gene in series in the same direction on the chromosome of the Pichia pastoris GS115 host strain for fusion and secretory expression. First, 4 copies of the BMP gene were obtained by artificially synthesizing the gene. And 6 consecutive histidines (6×His) were connected in series at the tail of 4 copies of the BMP gene in order to separate and purify the expressed target protein by using a metal chelate chromatography column, and then construct the expression 4 copies, 8 copies respectively on this basis. copy, 12 copy and 16 copy BMP gene engineering strains of Pichia pastoris, but the C-terminus introduced 6×His tag; Qian Wei et al. introduced formic acid and Based on the specific site of trypsin, the tandem monomers of the taste peptides were designed, and 8 copies of the genes of the three peptides were synthesized by splicing, cloned and expressed into the expression vector pET-30a and transformed into the host strain BL21(DE3 ), the target gene was successfully expressed after being induced by IPTG, and then digested. According to the charge difference, anion exchange chromatography was used to separate and purify the taste peptide monomer. The overall process is still cumbersome and difficult to purify.

At present, this technology has shortcomings such as difficulty in expression, low efficiency, and difficulty in recovery, and the expression of small molecular peptides often introduces specific affinity tags at the C-terminus, such as 6×His tag and other tag sequences cannot be effectively removed, resulting in damage to umami. Unavoidable negative effects of peptide taste

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