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Application of plb protein in constructing expression vector of fusion protein with chaperone-like function

A technology of fusion protein and expression vector, which is applied in the direction of introducing foreign genetic material, application, and fusion polypeptide by using the vector, which can solve problems such as differences, limited expression vectors of chaperone-like proteins, and unknown mechanism

Active Publication Date: 2022-03-08
江苏宇安医药研究有限公司
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  • 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 plb protein in constructing expression vector of fusion protein with chaperone-like function
  • Application of plb protein in constructing expression vector of fusion protein with chaperone-like function
  • Application of plb protein in constructing expression vector of fusion protein with chaperone-like function

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Example 1 Construction of the Escherichia coli expression vector comprising chaperone-like protein

[0061] Step 1. Gene synthesis and vector preparation of the PLB insert:

[0062] Select PLB1 as shown in SEQ ID: 1, carry out codon optimization of PLB1 encoding DNA, addition mutation and DNA sequence optimization of transcription initiation region, selection of flexible linker and codon optimization, blood coagulation factor Xa (FXa) recognition sequence amino acid Codon optimization, the specific steps are as follows:

[0063] (1) The amino acid sequence of PLB1 is reverse-translated into a DNA coding sequence, and its own coding sequence (SEQ ID NO: 6, 7) is selected through codon preference.

[0064] (2) Select the high-expression translation initiation region peptide sequence (MASTYKLILNGKTS), optimize its coding sequence and insert it into the 5'-end of PLB1 coding DNA (SEQ ID NO: 6-9).

[0065] (3) A flexible linker (KEKTPEEQL) between the PLB1 coding region an...

Embodiment 2

[0086] Example 2 Construction of pET28-PLB1 fusion protein expression vector test system

[0087] Step 1. Using pET28-PLB1 as the parent vector and hTTF3 as the target protein to test, construct the pET28-PLB1-hTFF3 fusion protein expression vector and expression strain:

[0088] 1. Artificially synthesized DNA sequence encoding hTFF3 mature peptide (SEQ ID NO: 28), DNA codons were optimized by DNAWorks software (SEQ ID NO: 29), and 6 primers for whole gene synthesis (SEQ ID NO: 31-36), The PCR whole gene synthesis method is the same as in Example 1 (see step 1, section (8)).

[0089] 2. The DNA restriction endonuclease sites required for recombination are introduced into the PCR whole gene synthesis primers: EcoR I (5'-end primer, SEQ ID NO: 31) and Xho I (3'-end primer, SEQ ID NO: 36), which is convenient for double enzyme digestion and recombination connection;

[0090] 3. The PCR product synthesized by the hTFF3 gene and the pET28-PLB1 vector were respectively digested w...

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Abstract

The invention discloses the application of a PLB protein in constructing a fusion protein expression carrier with a chaperone-like function, and successfully constructs a fusion protein expression carrier with a chaperone-like function by using the PLB protein. In the present invention, by constructing a pET-PLB1 expression vector containing a chaperone-like protein, a test target protein fusion protein expression vector, and a test target protein pET vector, through a comparative test of target protein expression, it is found that the soluble expression efficiency of the test target protein is improved, thereby It provides a useful tool for protein expression scientific research and industrial production.

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 PLB 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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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/31C12N15/62
CPCC12N15/70C07K14/315C07K2319/00
Inventor 李乾
Owner 江苏宇安医药研究有限公司
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