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Single chain antibody (single chain variable fragment, scFv) is formed by linking the variable regions of the heavy chain and light chain of an antibody through a flexible polypeptide
.
The molecular weight of scFv is only about 30 kDa, which has a high tissue penetration ability and is cleared faster than antibodies in vivo
.
scFv avoids immune responses mediated by the Fc region
.
These characteristics make scFv more suitable for application in the fields of in vivo imaging diagnosis, tumor and intracellular pathogen targeted therapy
.
.
The molecular weight of scFv is only about 30 kDa, which has a high tissue penetration ability and is cleared faster than antibodies in vivo
.
scFv avoids immune responses mediated by the Fc region
.
These characteristics make scFv more suitable for application in the fields of in vivo imaging diagnosis, tumor and intracellular pathogen targeted therapy
.
However, due to inherently low solubility and stability, only two E.
coli-produced scFvs have been approved for therapeutic use
.
Therefore, relevant means are urgently needed to improve the solubility and stability of scFv expressed in E.
coli, and provide a theoretical basis for the production, research and application of scFv
.
coli-produced scFvs have been approved for therapeutic use
.
Therefore, relevant means are urgently needed to improve the solubility and stability of scFv expressed in E.
coli, and provide a theoretical basis for the production, research and application of scFv
.
Recently, Associate Researcher Chen Shijie from the State Key Laboratory of New Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Chen Shijie, and Associate Researcher Wang Yongxiang from the Molecular Virology Education Department/Health Commission/Key Laboratory of the Academy of Medical Sciences, School of Basic Medicine, Fudan University formed a joint team.
Important progress has been made in the field of solubility and stability of single-chain antibody expressed in Escherichia coli.
The related result "A 33-residue peptide tag increases solubility and stability of Escherichia coli produced single-chain antibody fragments" was published online on August 8, 2022 in in the journal Nature Communications
.
Important progress has been made in the field of solubility and stability of single-chain antibody expressed in Escherichia coli.
The related result "A 33-residue peptide tag increases solubility and stability of Escherichia coli produced single-chain antibody fragments" was published online on August 8, 2022 in in the journal Nature Communications
.
The intrachain disulfide bonds of the heavy and light chain variable regions of scFvs are critical for their stability
.
The cytoplasm of common Escherichia coli not only lacks the components that catalyze the formation of disulfide bonds, but also has glutathione reductase and thioredoxin reductase to reduce disulfide bonds, so most of the scFv expressed in the cytoplasm of common Escherichia coli are Insoluble inclusion bodies
.
Some protein tags with solubilizing activity such as MBP, GST, NusA and Trx can further improve their solubility
.
However, due to the bulkiness of solubilizing tag proteins such as MBP, steric hindrance interferes with the binding of scFv to antigen, so it must be removed by proteolytic cleavage; and the removal of solubilizing tag proteins will make scFv unstable and induce inactivity.
aggregates
.
.
The cytoplasm of common Escherichia coli not only lacks the components that catalyze the formation of disulfide bonds, but also has glutathione reductase and thioredoxin reductase to reduce disulfide bonds, so most of the scFv expressed in the cytoplasm of common Escherichia coli are Insoluble inclusion bodies
.
Some protein tags with solubilizing activity such as MBP, GST, NusA and Trx can further improve their solubility
.
However, due to the bulkiness of solubilizing tag proteins such as MBP, steric hindrance interferes with the binding of scFv to antigen, so it must be removed by proteolytic cleavage; and the removal of solubilizing tag proteins will make scFv unstable and induce inactivity.
aggregates
.
Figure 1.
The P17 tag increases the solubility of the four scFvs in the E.
coli SHuffle strain
.
The P17 tag increases the solubility of the four scFvs in the E.
coli SHuffle strain
.
The P17 protein from the T7 phage tail specifically binds to the low-density lipoprotein receptor-related protein (LRP) on the surface of hepatocytes through a 33-amino acid polypeptide (called the P17 polypeptide or tag) that binds phage particles and small molecules, Targeted delivery of proteins, nucleic acids and liposomes to hepatocytes
.
The researchers chose G12-scFv, which can target hepatitis B virus envelope protein, as the research object.
In order to improve the efficiency of G12-scFv being endocytosed by hepatocytes, a P17 tag was fused to its carboxyl terminus
.
When expressed in E.
coli SHuffle strain, the solubility of G12-scFv-P17 protein was significantly higher than that of G12-scFv protein
.
The researchers found that the P17 tag could also effectively improve the solubility of the other three different scFvs in SHuffle strains by semi-quantitative solubility assay
.
Through deletion mutation and point mutation analysis, it was found that the solubilization effect of P17 tag on scFv was related to its hydrophilic sequence, especially the charged amino acid residues, but not to the possible α-helix secondary structure
.
.
The researchers chose G12-scFv, which can target hepatitis B virus envelope protein, as the research object.
In order to improve the efficiency of G12-scFv being endocytosed by hepatocytes, a P17 tag was fused to its carboxyl terminus
.
When expressed in E.
coli SHuffle strain, the solubility of G12-scFv-P17 protein was significantly higher than that of G12-scFv protein
.
The researchers found that the P17 tag could also effectively improve the solubility of the other three different scFvs in SHuffle strains by semi-quantitative solubility assay
.
Through deletion mutation and point mutation analysis, it was found that the solubilization effect of P17 tag on scFv was related to its hydrophilic sequence, especially the charged amino acid residues, but not to the possible α-helix secondary structure
.
Figure 2.
P17 tag enhances the thermal stability of G12-scFv
.
P17 tag enhances the thermal stability of G12-scFv
.
Through thermal stability experiments, the research team found that the P17 tag, like the intrachain disulfide bond, enhanced the thermal stability of the scFv
.
Furthermore, the antigen-binding ability of the P17-tag fused G12-scFv and its virus-neutralizing activity were more than two-fold enhanced, suggesting that the P17 tag has a type I intramolecular chaperone-like activity
.
In conclusion, this study found that the 33-peptide tag can significantly improve the solubility and stability of single-chain antibodies in E.
coli Shuffle cells, providing a theoretical basis for the production, research and application of single-chain antibodies
.
.
Furthermore, the antigen-binding ability of the P17-tag fused G12-scFv and its virus-neutralizing activity were more than two-fold enhanced, suggesting that the P17 tag has a type I intramolecular chaperone-like activity
.
In conclusion, this study found that the 33-peptide tag can significantly improve the solubility and stability of single-chain antibodies in E.
coli Shuffle cells, providing a theoretical basis for the production, research and application of single-chain antibodies
.
The corresponding authors of the paper are Associate Researcher Wang Yongxiang of Fudan University and Associate Researcher Chen Shijie of Luo Cheng's research group of Shanghai Institute of Materia Medica.
The first authors are Wang Yang and Yuan Wenjie, master graduates of Fudan University, and Guo Siqi, a doctoral student jointly trained by Shanghai Institute of Materia Medica/Nanchang University.
.
The research was also supported and helped by Academician Wen Yumei of Fudan University, Researcher Yuan Zhenghong, Professor Tong Shuping, Researcher Chen Zhenguo, Researcher Luo Cheng of Shanghai Institute of Materia Medica, and Professor Michael Nassal of the University of Freiburg, Germany
.
This work was supported by the National Natural Science Foundation of China and the Shanghai Science and Technology Commission project
.
The first authors are Wang Yang and Yuan Wenjie, master graduates of Fudan University, and Guo Siqi, a doctoral student jointly trained by Shanghai Institute of Materia Medica/Nanchang University.
.
The research was also supported and helped by Academician Wen Yumei of Fudan University, Researcher Yuan Zhenghong, Professor Tong Shuping, Researcher Chen Zhenguo, Researcher Luo Cheng of Shanghai Institute of Materia Medica, and Professor Michael Nassal of the University of Freiburg, Germany
.
This work was supported by the National Natural Science Foundation of China and the Shanghai Science and Technology Commission project
.
Full text link: by: Luo Cheng's research group)
