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Application of PGB protein in construction of fusion protein expression vector with chaperone-like protein effect

A technology for expressing vectors and fusion proteins, which is applied in the direction of expression enhancement of stability/folding protein fusion, introduction of foreign genetic material and depsipeptides using vectors, etc., which can solve the problems of limited expression vectors, differences, and unknown mechanisms of chaperone-like proteins.

Inactive Publication Date: 2019-12-10
因之彩生物科技(武汉)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing chaperone-like protein expression vectors are very limited, and there are certain differences in the auxiliary folding effect on different target proteins, and the mechanism is unclear. Therefore, it is necessary to further increase the selectivity of chaperone-like protein expression vectors

Method used

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  • Application of PGB protein in construction of fusion protein expression vector with chaperone-like protein effect
  • Application of PGB protein in construction of fusion protein expression vector with chaperone-like protein effect
  • Application of PGB protein in construction of fusion protein expression vector with chaperone-like protein effect

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Construction of the Escherichia coli expression vector comprising chaperone-like protein

[0067] Step 1. Gene synthesis and vector preparation of the PGB insert:

[0068] Taking PGB3 as shown in SEQ ID: 3 as an example, the codon optimization of PGB3 encoding DNA, the addition mutation of the transcription initiation region and DNA sequence optimization, the selection of flexible linkers and codon optimization, and the recognition of blood coagulation factor Xa (FXa) Sequence amino acid codon optimization, the specific steps are as follows:

[0069] (1) The amino acid sequence of PGB3 is reverse-translated into a DNA coding sequence, and its own coding sequence (SEQ ID NO: 4, 5) is selected through codon preference.

[0070] (2) Add start codon and adapter sequence [MAS], and insert into the N-terminal of PGB3 protein and the 5'-terminal of coding DNA (SEQ ID NO: 4-7).

[0071] (3) Select a flexible linker between the PGB3 coding region and the PBS coding r...

Embodiment 2

[0092] Example 2 Construction of pET28-PGB3 fusion protein expression vector test system

[0093] Step 1. Using pET28-PGB3 as the parent vector and hTFF2 as the test target protein, construct the pET28-PGB3-hTFF2 fusion protein expression vector and expression strain:

[0094] 1. Artificially synthesized DNA sequence encoding hTFF2 mature peptide (SEQ ID NO: 25), the DNA codon was optimized by DNAWorks software (SEQ ID NO: 26), and 8 primers were synthesized for the whole gene (SEQ ID NO: 27-34, PCR The DNA restriction endonuclease sites required for recombination are introduced into the primers for the whole gene synthesis: EcoR I (5'-end primer, SEQ ID NO: 27) and Xho I (3'-end primer, SEQ ID NO: 34 ), to facilitate double-enzyme digestion and recombination insertion.

[0095] 2. The hTFF2 PCR whole gene synthesis method is the same as in Example 1 (see step 1, section (8)).

[0096] 3. The PCR product synthesized by the hTFF2 gene and the pET28-PGB3 vector were digested w...

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Abstract

The invention discloses application of a PGB protein in construction of a fusion protein expression vector with a chaperone-like protein effect. The fusion protein expression vector with the chaperone-like protein effect is successfully constructed by utilizing the PGB protein. For the application, by constructing a v chaperone-like protein contained pET-PGB3 expression vector and testing a targetprotein fusion protein expression vector and a target protein contained pET control vector, through contrast tests of target protein induction expressions in different recombinants, discovered that PGB has a novel chaperone-like protein function for improving the soluble expression efficiency of the target protein, so that a useful tool is provided to scientific research and industrial productionof protein expression.

Description

technical field [0001] The present invention relates to the technical field of genetic engineering and protein engineering, in particular to the technical field of protein fusion expression, in particular to the application of a PGB protein in constructing a fusion protein expression vector with chaperone-like protein function. Background technique [0002] The problem of protein folding is listed as an important topic of "biophysics in the 21st century". It is a major biological problem that has not yet been resolved by the central dogma of molecular biology. In protein expression (production) engineering, obtaining natural structural protein is a prerequisite for soluble protein expression, the basis for ensuring the physiological function of the target protein, and a cost-saving link in the industrial protein production process. [0003] The function of native proteins depends on the physiological conformation of the protein. Molecular biochemical science believes that t...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/62C07K14/00
CPCC07K14/00C07K2319/35C12N15/62C12N15/63
Inventor 李乾
Owner 因之彩生物科技(武汉)有限公司
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