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. 2023 Apr;65(4):581-589.
doi: 10.1007/s12033-022-00561-6. Epub 2022 Sep 12.

Application of an Autoinduction Strategy to Optimize the Heterologous Production of an Antitumor Bispecific Fusion Protein Based on the TRAIL Receptor-Selective Mutant Variant in Escherichia coli

Alina Isakova  1 , Artem Artykov  1 , Yekaterina Vorontsova  1 , Dmitry Dolgikh  1   2 , Mikhail Kirpichnikov  1   2 , Marine Gasparian  1 , Anne Yagolovich  3   4
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Application of an Autoinduction Strategy to Optimize the Heterologous Production of an Antitumor Bispecific Fusion Protein Based on the TRAIL Receptor-Selective Mutant Variant in Escherichia coli

Alina Isakova et al. Mol Biotechnol. 2023 Apr.

Abstract

Autoinduction is a simple approach for heterologous protein expression that helps to achieve the high-level production of recombinant proteins in soluble form. In this work, we investigated if the application of an autoinduction strategy could help to optimize the production of bifunctional protein SRH-DR5-B, the DR5-specific TRAIL variant DR5-B fused to a VEGFR2-specific peptide SRHTKQRHTALH for dual antitumor and antiangiogenic activity. The protein was expressed in Escherichia coli SHuffle B T7, BL21(DE3), and BL21(DE3)pLysS strains. By IPTG induction, the highest expression level was in SHuffle B T7, while by autoinduction, the similar expression level was achieved in BL21(DE3)pLysS. However, in SHuffle B T7, only 45% of IPTG-induced SRH-DR5-B was expressed in soluble form, in contrast to 75% autoinduced in BL21(DE3)pLysS. The yield of purified SRH-DR5-B protein expressed by autoinduction in BL21(DE3)pLysS was 28 ± 4.5 mg per 200 ml of cell culture, which was 1.4 times higher than the yield from IPTG-induced SHuffle B T7. Regardless of the production method, SRH-DR5-B was equally cytotoxic to BxPC-3 human tumor cells expressing DR5 and VEGFR2 receptors. Thus, the production of SRH-DR5-B by autoinduction in the E. coli BL21(DE3)pLysS strain is an efficient, technologically simple, and economical technique that allows to obtain a large amount of active protein from the cytoplasmic cell fraction. Our work demonstrates that the strategy of induction of protein expression is no less important than the strain selection.

Keywords: Autoinduction; DR5-B; E. coli; Protein expression; TRAIL; VEGFR2.

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