Method for simultaneously enhancing expression quantity and solubility of target protein in prokaryotic system

A protein expression and solubility technology, applied in the biological field, can solve the problems of unpredictable target protein interaction and reduced work efficiency

Active Publication Date: 2022-05-13
SHANXI AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is currently not possible to predict which protein of interest interacts with a specific chaperone and produces a solubilizing effect
Usually, it is necessary to try one by one, such as the Chaperone Plasmid Set of Japan Takara Company, which also provides a co-expression solution for multiple molecular chaperones, allowing users one by one, reducing work efficiency

Method used

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  • Method for simultaneously enhancing expression quantity and solubility of target protein in prokaryotic system
  • Method for simultaneously enhancing expression quantity and solubility of target protein in prokaryotic system
  • Method for simultaneously enhancing expression quantity and solubility of target protein in prokaryotic system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Construction of fusion protein VeGFP

[0035] This example exemplifies a fusion protein VeGFP spliced ​​with the N-terminal signal peptide of the naturally occurring protein Vip3Aa (SEQ ID NO: 1) and eGFP protein.

[0036] The characteristics of the VeGFP protein are: the N-terminal signal peptide sequence of Vip3Aa is located at the N-terminal of the eGFP protein; there are no other redundant amino acids between the N-terminal signal peptide of Vip3Aa (SEQ ID NO: 1) and the amino acid sequence of the eGFP protein. The polynucleotide sequence encoding the VeGFP protein can be obtained from at least three approaches: 1) by obtaining the polynucleotide sequence of the N-terminal signal peptide encoding Vip3Aa and the amino acid sequence of the eGFP protein with a polymerase chain reaction (PCR reaction), and then The two are spliced ​​together by overlapping PCR (Horton, R.M.et al.BioTechniques, 1990) to form a complete fusion gene; 2) by combining the N-ter...

Embodiment 2

[0037] Example 2: Construction of the prokaryotic cell expression vector p28aD-VeGFP of the fusion protein VeGFP

[0038] This example illustrates a prokaryotic expression vector for the fusion protein described in Example 1 herein. First, the two ends of the VeGFP gene were added to BamHI (5') and SacI (3') sites respectively. Then the gene was inserted into the corresponding site of pET28aDel vector (Gao et al.2011) to form p28aD-VeGFP expression vector ( figure 1 ). As a control, the two ends of the eGFP gene were added to the BamHI (5′) and SacI (3′) sites to form the p28aD-eGFP expression vector ( figure 1 ). The two plasmids were transformed into Escherichia coli BL21(DE3) star strain to obtain BL28-VeGFP and BL28-eGFP strains, respectively.

Embodiment 3

[0039] Embodiment 3: Expression and comparison of VeGFP protein and eGFP protein

[0040] The BL28-VeGFP and BL28-eGFP strains were respectively streaked in solid LB medium (Luria-Bertani medium) containing 50 μg / mL kanamycin, and cultured overnight at 37°C. Select 6 single clones from each strain, pick them up and insert them into the liquid LB medium containing 50 μg / mL kanamycin, shake culture (37° C., 200 rpm) to OD600=0.8. IPTG (isopropylβ-D-1-thiogalactopyranoside) was added to the medium at a final concentration of 1 mmol / L. Under the condition of 16°C, the expression was induced for 16 hours. After induction of expression, pipette 500 μL of each tube of bacterial liquid into a new 1.5 mL centrifuge tube, centrifuge at 12,000 rpm for 5 min, and collect bacterial cells. With 200μL PBS buffer (137mM NaCl, 2.7mM KCl, 10mMNa 2 HPO 4 , and 2mM KH 2 PO 4 , pH 7.4) to resuspend the cells, and add 50 μL of 5× protein loading buffer (250 mM Tris (pH 6.8), 10% (w / v) SDS, 0....

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Abstract

The invention discloses a method for simultaneously enhancing the expression quantity and solubility of a target protein in a prokaryotic system, and the method is used for guiding the expression of the target protein by using a novel fusion tag based on a Vip3 protein signal peptide sequence or a local sequence of the Vip3 protein signal peptide sequence. A fusion gene formed by the fusion tag and a coding gene of a target protein is co-expressed with an escherichia coli molecular chaperone Trigger factor gene or a homologous gene thereof in other organisms in the same prokaryotic expression system, so that the expression quantity and the solubility of the target protein can be enhanced. The importance of the invention lies in that a novel fusion tag is invented, the expression quantity of the target protein can be remarkably enhanced, and meanwhile, the tag can interact with a known specific molecular chaperone, so that the solubility of the target protein can be foreseeably enhanced on the premise of ensuring the expression quantity, one-by-one attempts on different molecular chaperones are avoided, and the target protein solubility is improved. The traditional scheme for enhancing the solubility of the unknown target protein can be determined, and the efficiency is greatly improved.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for simultaneously enhancing the expression and solubility of a target protein in a prokaryotic system. Background technique [0002] When using prokaryotic system to express recombinant target protein, in order to increase the solubility of the target protein and prevent the formation of inclusion bodies, a variety of soluble tags can be used to regulate, such as MBP (maltose-binding protein) (diGuan etal.1988; Kapust and Waugh, 1999) Label, GST (glutathione-S-transferase) (Smithand Johnson, 1988) label, SUMO (small ubiquitin related modifier) ​​(Butt et al.2005; Marblestone et al.2006), NusA (N-utilization substance) (Davis et al .1999) label and Trx (Thioredoxin) (Lavallie et al.1993) and so on. After this type of tag is attached to the N-terminal of the target protein, it can increase the solubility of the target protein, thereby making it easier to form the correct spa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/67C07K14/32C12R1/19
CPCC12N15/70C12N15/67C07K14/32C07K2319/02C07K2319/35Y02A50/30
Inventor 高建华欧阳春平赵娟丽王兴春
Owner SHANXI AGRI UNIV
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