A single nucleotide mutation drastically increases the expression of tumor-homing NGR-TNFα in the E. coli M15-pQE30 system by improving gene transcription
| Autor: | Hao Yang, Xiaofeng Lu, Ze Tao, Jie Fan, Zhao Li, Shengfu Li, Youmei Jin, Qiuxiao Shi, Yanru Feng, Jie Chen, Jingqiu Cheng |
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| Rok vydání: | 2021 |
| Předmět: |
Transcription
Genetic Tumor vasculature normalization Galanin Substance P medicine.disease_cause Applied Microbiology and Biotechnology 03 medical and health sciences Recombinant expression Valine Transcription (biology) Cell Line Tumor Escherichia coli medicine Gene 030304 developmental biology chemistry.chemical_classification 0303 health sciences Mutation Oligopeptide Tumor necrosis factor alpha Nucleotides Tumor Necrosis Factor-alpha 030306 microbiology General Medicine Nucleotide mutation Molecular biology Biotechnological Products and Process Engineering Amino acid chemistry Leucine Oligopeptides Biotechnology |
| Zdroj: | Applied Microbiology and Biotechnology |
| ISSN: | 1432-0614 0175-7598 |
| DOI: | 10.1007/s00253-021-11136-x |
| Popis: | Due to their potent immune stimulation, tumor necrosis factor alpha (TNFα) variants with tumor-homing activity are attractive as novel antitumor drugs. The promising antitumor effect of NGR-TNFα in clinical trials triggered extensive interest in developing novel tumor-homing TNFα variants in recent years. Owing to its promising antitumor effect, NGR-TNFα is usually used as a control for newly developed tumor-homing TNFα variants. In our previous works, we produced a pericyte-targeting Z-TNFα at high levels using the Escherichia coli (E. coli) M15-pQE30 system. To further compare Z-TNFα and NGR-TNFα, we attempted to express NGR-TNFα using the same system. Surprisingly, native NGR-TNFα was expressed at a low (~ 0.2 mg/L) level in E. coli M15 containing the pQE30 plasmid. However, a single nucleotide mutation of C to G, resulting in a substitution of leucine (L) with valine (V) at the start of TNFα, increased the expression of NGR-TNFα by ~ 100 times through improving transcription. In addition, the amino acid substitution showed a little impact on the receptor binding, in vitro cytotoxicity, and in vivo antitumor effect of NGR-TNFα. As fusing NGR to the N-terminus of TNFα with a valine substitution did not reduce the protein yield, the TNFα gene with a C > G mutation might be used to prepare novel tumor-homing TNFα when the native TNFα-based variant is expressed at an extremely low level in E. coli. Notably, in addition to the mutated valine, the impact of N-terminal additional amino acids provided by pQE30 vector on the function of TNFα variant must be carefully evaluated. Key points • A single nucleotide mutation increased the expression of NGR-TNFα by two orders. • Nucleotide mutation-induced amino acid substitution did not reduce NGR-TNFα activity. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-021-11136-x. |
| Databáze: | OpenAIRE |
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