Optimized Heterologous Expression and Efficient Purification of a New TRAIL-Based Antitumor Fusion Protein SRH–DR5-B with Dual VEGFR2 and DR5 Receptor Specificity

Yagolovich, Anne V.; Artykov, Artem A.; Isakova, Alina A.; Vorontsova, Yekaterina V.; Dolgikh, Dmitry A.; Kirpichnikov, Mikhail P.; Gasparian, Marine E.

doi:10.3390/ijms23115860

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Optimized Heterologous Expression and Efficient Purification of a New TRAIL-Based Antitumor Fusion Protein SRH–DR5-B with Dual VEGFR2 and DR5 Receptor Specificity

by

Anne V. Yagolovich

^1,2,*

,

Artem A. Artykov

¹

,

Alina A. Isakova

¹,

Yekaterina V. Vorontsova

¹,

Dmitry A. Dolgikh

^1,2

,

Mikhail P. Kirpichnikov

^1,2 and

Marine E. Gasparian

^1,*

¹

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, 117997 Moscow, Russia

²

Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

^*

Authors to whom correspondence should be addressed.

Int. J. Mol. Sci. 2022, 23(11), 5860; https://doi.org/10.3390/ijms23115860

Submission received: 27 April 2022 / Revised: 17 May 2022 / Accepted: 20 May 2022 / Published: 24 May 2022

(This article belongs to the Special Issue Heterologous Expression of Difficult to Produce Proteins in Bacterial Systems 2.0)

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Abstract

In the last two decades, bifunctional proteins have been created by genetic and protein engineering methods to increase therapeutic effects in various diseases, including cancer. Unlike conventional small molecule or monotargeted drugs, bifunctional proteins have increased biological activity while maintaining low systemic toxicity. The recombinant anti-cancer cytokine TRAIL has shown a limited therapeutic effect in clinical trials. To enhance the efficacy of TRAIL, we designed the HRH–DR5-B fusion protein based on the DR5-selective mutant variant of TRAIL fused to the anti-angiogenic synthetic peptide HRHTKQRHTALH. Initially low expression of HRH–DR5-B was enhanced by the substitution of E. coli-optimized codons with AT-rich codons in the DNA sequence encoding the first 7 amino acid residues of the HRH peptide. However, the HRH–DR5-B degraded during purification to form two adjacent protein bands on the SDS-PAGE gel. The replacement of His by Ser at position P2 immediately after the initiator Met dramatically minimized degradation, allowing more than 20 mg of protein to be obtained from 200 mL of cell culture. The resulting SRH–DR5-B fusion bound the VEGFR2 and DR5 receptors with high affinity and showed increased cytotoxic activity in 3D multicellular tumor spheroids. SRH–DR5-B can be considered as a promising candidate for therapeutic applications.

Keywords: bifunctional fusion proteins; fusion expression; E. coli; TRAIL DR5-selective variant DR5-B; DR5 receptor; VEGFR2 receptor

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MDPI and ACS Style

Yagolovich, A.V.; Artykov, A.A.; Isakova, A.A.; Vorontsova, Y.V.; Dolgikh, D.A.; Kirpichnikov, M.P.; Gasparian, M.E. Optimized Heterologous Expression and Efficient Purification of a New TRAIL-Based Antitumor Fusion Protein SRH–DR5-B with Dual VEGFR2 and DR5 Receptor Specificity. Int. J. Mol. Sci. 2022, 23, 5860. https://doi.org/10.3390/ijms23115860

AMA Style

Yagolovich AV, Artykov AA, Isakova AA, Vorontsova YV, Dolgikh DA, Kirpichnikov MP, Gasparian ME. Optimized Heterologous Expression and Efficient Purification of a New TRAIL-Based Antitumor Fusion Protein SRH–DR5-B with Dual VEGFR2 and DR5 Receptor Specificity. International Journal of Molecular Sciences. 2022; 23(11):5860. https://doi.org/10.3390/ijms23115860

Chicago/Turabian Style

Yagolovich, Anne V., Artem A. Artykov, Alina A. Isakova, Yekaterina V. Vorontsova, Dmitry A. Dolgikh, Mikhail P. Kirpichnikov, and Marine E. Gasparian. 2022. "Optimized Heterologous Expression and Efficient Purification of a New TRAIL-Based Antitumor Fusion Protein SRH–DR5-B with Dual VEGFR2 and DR5 Receptor Specificity" International Journal of Molecular Sciences 23, no. 11: 5860. https://doi.org/10.3390/ijms23115860

APA Style

Yagolovich, A. V., Artykov, A. A., Isakova, A. A., Vorontsova, Y. V., Dolgikh, D. A., Kirpichnikov, M. P., & Gasparian, M. E. (2022). Optimized Heterologous Expression and Efficient Purification of a New TRAIL-Based Antitumor Fusion Protein SRH–DR5-B with Dual VEGFR2 and DR5 Receptor Specificity. International Journal of Molecular Sciences, 23(11), 5860. https://doi.org/10.3390/ijms23115860

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Optimized Heterologous Expression and Efficient Purification of a New TRAIL-Based Antitumor Fusion Protein SRH–DR5-B with Dual VEGFR2 and DR5 Receptor Specificity

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