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Auxiliary protein for producing recombinant fusion protein, coding gene, recombinant fusion protein, recombinant expression vector and preparation method

A technology of fusion protein and auxiliary protein, which is applied in the field of fusion protein, can solve the problems of protein insolubility and high production cost of inclusion bodies, and achieve the effects of good thermal stability, cost reduction and simplified extraction process

Inactive Publication Date: 2018-11-30
CHENGDU ENZPRO BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems in the prior art that the above-mentioned produced protein is insoluble in the supernatant and the production cost is high by using inclusion bodies, the present invention provides an auxiliary protein, coding gene, recombinant fusion protein, recombinant expression Vector and preparation method of the recombinant fusion protein

Method used

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  • Auxiliary protein for producing recombinant fusion protein, coding gene, recombinant fusion protein, recombinant expression vector and preparation method
  • Auxiliary protein for producing recombinant fusion protein, coding gene, recombinant fusion protein, recombinant expression vector and preparation method
  • Auxiliary protein for producing recombinant fusion protein, coding gene, recombinant fusion protein, recombinant expression vector and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The purpose of this example is to provide three auxiliary proteins (Thioredoxin) and a recombinant fusion protein comprising the three auxiliary proteins.

[0030] According to the amino acid sequence (serial number: KFL15614.1) of Thioredoxin published online, the cysteine ​​at position 29 of this Thioredoxin is mutated to serine and the last amino acid is deleted, such as Seq Shown in ID No.3; on the basis of Seq ID No.3, three amino acids (GSG) or five amino acids (GSGSG) are added at its C-terminus to increase the flexibility of the recombinant fusion protein, and its amino acid sequences are as Seq ID No.1 and Seq ID No.2; on the basis of Seq ID No.1-3, connect the enzyme cleavage site of enterokinase and glucagon-1 analog 7-36 at the C-terminus in sequence , the amino acid sequences of which are respectively shown in Seq ID No.7, Seq ID No.9 and Seq ID No.11. According to the aforementioned amino acid sequence, its nucleotide sequence is reversed, and the reverse...

Embodiment 2

[0036] The purpose of this example is to construct a recombinant expression vector.

[0037] Insert the nucleotide sequence of the gene encoding the above auxiliary protein into the NcoI (CCATGG) and XhoI (CTCGAG) multiple cloning sites of the vector pET28a respectively to obtain a recombinant expression vector, sequence the constructed recombinant expression vector, and verify that the inserted gene is correct , named pET28a-mtrA / 7-36, pET28a-mtrA1 / 7-36 and pET28a-mtrA2 / 7-36. In addition to pET28a, other vectors of pET series vectors can also be used, such as pET-28b, pET-28c, pET-29a, pET-30a, pET-30b, pET-30c, pET-33b, pET-39b, pET-40b, pET-41a, pET-41b, pET-41c, pET-42a, pET-42b, pET-42c, pET-47b, pET-48b, pET-49b, pET-50b, pET-51 or pET-52b. The recombinant fusion proteins inserted in the three recombinant expression vectors specifically include the following three types:

[0038] (1) pET28a-mtrA / 7-36: The amino acid sequence of the recombinant fusion protein of the recom...

Embodiment 3

[0042] The purpose of this example is to prepare recombinant engineering bacteria.

[0043] The correctly constructed expression vectors pET28a-mtrA / 7-36, pET28a-mtrA1 / 7-36 and pET28a-mtrA2 / 7-36 were respectively heat-shock transformed into E. Kanamycin LB plate medium. For the preparation of Escherichia coli BL21(DE3) and the method of heat shock transformation, please refer to the "Molecular Cloning Experiment Guide". After the transformants grow out, select a few and send them for sequencing. The transformants with correct sequencing are stored for future use, and the transformants with correct sequencing are recombinant engineering bacteria.

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Abstract

The invention belongs to the technical field of fusion proteins, and discloses an auxiliary protein which is a protein with an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3,or a protein which has homology of 85% or more with the protein as shown in the SEQ ID NO.1. The invention also discloses a recombinant fusion protein comprising the auxiliary protein, a recombinant expression vector and a preparation method of the recombinant fusion protein. The method has the advantages that the auxiliary protein can be used for producing a plurality of micro-molecule polypeptides which has amino acids with a number of 20-80 and / or has an isoelectric point range of 3-9. The recombinant fusion protein can be secreted out of a host cell, can stably exist in a supernate at a temperature of 4-100 DEG C and has relatively good thermal stability. A large amount of the recombinant fusion proteins can be obtained at a temperature of 60-100 DEG C through a thermal wall-breaking method, the extraction process of the recombinant fusion protein is simplified, and the cost is reduced. Then, the auxiliary protein is broken from a target polypeptide through enzyme cutting or chemical cutting under appropriate conditions, so that a stable and active target polypeptide can be obtained.

Description

technical field [0001] The invention belongs to the technical field of fusion proteins, and in particular relates to an auxiliary protein, a coding gene, a recombinant fusion protein, a recombinant expression vector and a preparation method for producing a recombinant fusion protein. Background technique [0002] Many kinds of polypeptides and proteins can be expressed by expression vectors, transformed microorganisms or animal or plant host cells comprising the nucleotide sequence encoding the polypeptides. There are many ways to express the target polypeptide, one is to directly secrete the target polypeptide outside the cell, the other is to express it directly from the natural N-terminus of the target polypeptide in the cell, and another method is to express the target polypeptide at the N-terminus Or add an auxiliary protein sequence to the C-terminus so that the auxiliary protein and the target polypeptide can be co-expressed. [0003] The Escherichia coli expression ...

Claims

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Application Information

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IPC IPC(8): C07K14/47C12N15/12C07K19/00C12N15/62C12N15/70C12N1/21C12R1/19
CPCC07K7/14C07K14/47C07K14/605C07K14/61C07K2319/50C12N15/70
Inventor 梁莉刘懿
Owner CHENGDU ENZPRO BIOTECHNOLOGY CO LTD