Method for modeling production of fusion protein by utilizing trans-splicing of intein

Abstract

The invention discloses a method for the modeling production of fusion protein by utilizing the trans-splicing of intein, which can be used in fields, such as gene engineering, enzyme production, medicine production, food industry, farming and animal industry, and the like. In the invention, A target protein part with special biological functions and auxiliary protein with general functions are mainly connected together by utilizing the trans-splicing of the intein so as to obtain the fusion protein; the target protein T, the auxiliary protein P1 and P2, the intein A and the intein B which are mutually incompatible (such as blue algae: DnaE and DnaB of Synechocystis sp. PCC6803) are selected; the intein A and intein B are respectively decomposed into an amino terminal (N terminal) and a carboxyl terminal (C terminal), i.e. An, Ac, Bn and Bc; after different carrier systems are constructed; protein segments Ac-T-Bn, P1-An, Bc-P2, An and Bc are respectively obtained through host expression; the obtained protein segments are mixed; and the trans-splicing of the intein is carried out to generate the fusion protein P1-T-P2, P1-T or T-P2.

Description

technical field [0001] The invention relates to a method for patterned production of recombinant fusion protein, which can be used in the fields of genetic engineering, enzyme production, pharmaceutical production, food industry, agriculture and animal husbandry and the like. Background technique [0002] 1. Intein-mediated trans-splicing principle [0003] Intein (intein) is a polypeptide chain parasitic in other proteins. Its coding DNA sequence is transcribed and translated together with the exons of the normal protein to produce a polypeptide chain. Then cut out the sequence corresponding to the intein, and then connect the sequences of the parasitized protein to become a functional protein. This is a protein self-splicing mechanism. Generally speaking, intein has 10 motifs (motif), the order is N'-AN 2 BN 4 CDEHFG-C', where A, N 2 , B, N 4 , F, and G belong to the motif of the self-splicing domain, while C, D, E, and H belong to the motif of the homing endonuclea...

AI Technical Summary

Problems solved by technology

It has the following disadvantages: 1) for the combination of the same target protein and multiple auxiliary proteins, multiple fusion genes will be synthesized, which increases a lot of workload; , such as vector construction, transformation, expression and purification, etc. have brought more problems; 3) fusion genes are limited to expression in a single host organism, and the two parts of the fusion protein often require different host expression environments

For the production of multiple fusion proteins of the same target protein, the chemical fusion method is much simpler than the gene fusion method, but it also has very large defects: 1) The specificity of the chemical fusion method is very poor, and the integration sites of the two proteins are uncertain , and sometimes lead to the fusion of the target protein and the auxiliary protein, so the final fusion protein yield is very low, and separation and purification are also difficult; 2) Some auxiliary proteins, such as His-tag composed of 6 amino acids, are very Short, can not be effectively produced by genetic engineering, can only be produced by chemical synthesis, and the production cost is very high; 3) The chemical cross-linked substances used in the chemical fusion method are often toxic and harmful substances, so it poses high demands on the production environment 4) The purity of the target protein and auxiliary protein has very high requirements, and two purification steps are added to increase the production cost

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