Intestinal trefoil factor recombinant expression vector and preparation method of intestinal trefoil factor

Abstract

The invention discloses a process which uses pGAP as a promoter and Pichia Yeast GS115 as an engineering bacterium to perform fermentation expression on an intestinal trefoil factor (ITF) in a fermentation tank. The method comprises the following steps: the recombinant Pichia Yeast expression vector pGAPZ alpha A-ITF is firstly constructed and then recombined in Escherichia coli Top10 to amplify, Escherichia coli plasmid is extracted and linearized to be transformed in Saccharomyce GS115, a high copy transformant is screened according to the zeocin resistance gene carried by the vector to perform constitutive expression, a high expression pilot test engineering bacterium is screened and fermented in a culture medium for 2 days to perform secretory expression on the ITF; and the target protein ITF of which the purity is more than 95% is obtained through purification treatment, wherein the output is about 200mg / L and the yield is about 50%. The intestinal trefoil factor recombinant expression vector has low production cost, short fermentation period and simple purification method; methanol induction is not adopted, the protein expression level is high; and a good foundation is laid for the large-scale industrial production of ITF.

Description

technical field [0001] The invention belongs to the field of biotechnology and engineering, in particular to a preparation method of intestinal trefoil factor. Background technique [0002] Intestinal trefoil factor (ITF for short) is a specific small molecule protein distributed in the intestinal tract, with a molecular weight of about 6-7kD. ITF consists of 59 amino acid residues, including a specific sequence consisting of 38-39 amino acid residues, in which 6 cysteines form 3 cysteines in the order of 1-5, 2-4, 3-6. Disulfide bonds, resulting in a specific and stable three-lobed structure. This stable structure ensures its activity in the gut, independent of digestive enzymes and pH changes. Under normal circumstances, ITF is distributed throughout the small intestine, especially at the base of the small intestinal villous ring cells. ITF exists in the form of 20% monomer and 80% dimer in vivo, but only the dimer has biological activity. ITF can not only promote the ...

AI Technical Summary

Problems solved by technology

But its defect is: when it is expanded from the laboratory to the industrial scale, its output declines rapidly, because the selection pressure of the high copy number of the Werther plasmid in the medium disappears, the plasmid becomes unstable, and the copy number decreases, resulting in a decrease in the output. The reduction is only 48mg / L; its purification method is cumbersome, complicated operation and many steps

However, according to the method it introduces, it has the following disadvantages: 1. The intestinal trefoil factor is obtained by expressing eukaryotic cells. Although the protein activity is still good, the cost is high, the yield is low, and it is difficult to produce on a large scale; 2. The intestinal mucosa is used to extract the intestinal trefoil factor. Leaf factor, although natural intestinal trefoil factor can be obtained, its source of raw material (intestinal mucosa) is limited, the yield is very low, and it cannot be used for large-scale industrial production

Images