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A scalable insect cell-based production process of the human recombinant BMX for in-vitro covalent ligand high-throughput screening

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Abstract

Bone Marrow Tyrosine kinase in the chromosome X (BMX) is a TEC family kinase associated with numerous pathological pathways in cancer cells. Covalent inhibition of BMX activity holds promise as a therapeutic approach against cancer. To screen for potent and selective covalent BMX inhibitors, large quantities of highly pure BMX are normally required which is challenging with the currently available production and purification processes. Here, we developed a scalable production process for the human recombinant BMX (hrBMX) using the insect cell-baculovirus expression vector system. Comparable expression levels were obtained in small-scale shake flasks (13 mL) and in stirred-tank bioreactors (STB, 5 L). A two-step chromatographic-based process was implemented, reducing purification times by 75% when compared to traditional processes, while maintaining hrBMX stability. The final production yield was 24 mg of purified hrBMX per litter of cell culture, with a purity of > 99%. Product quality was assessed and confirmed through a series of biochemical and biophysical assays, including circular dichroism and dynamic light scattering. Overall, the platform herein developed was capable of generating 100 mg purified hrBMX from 5 L STB in just 34 days, thus having the potential to assist in-vitro covalent ligand high-throughput screening for BMX activity inhibition.

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Acknowledgments

This work has received funding from the European Union Horizon 2020 research and innovation program, under grant agreement No 702428. Royal Society to G.J.L.B. (URF\R\180019), FCT Portugal (iFCT to G.J.L.B., IF/00624/2015, Postdoctoral Fellowship SFRH/BPD/95253/2013 to J.D.S., and PTDC/MED-QUI/28764/2017 to J.D.S.), Marie Sklodowska-Curie IF (grant agreement No. 702428 to J.D.S. Investigador FCT programme 2014 (IF/01704/2014 and IF/01704/2014/CP1229/CT0001) to A.R. and from iNOVA4 Health Research Unit (LISBOA-01-0145-FEDER-007344) FCT/ Ministério da Educação e Ciência, FCT/MCTES, FEDER, and Programa Operacional Competitividade e Internacionalização (POCI) are also acknowledged. FCT SFRH/BPD/118731/2016 to M.C.M. FCT SFRH/BD/143583/2019 to B.B.S.

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Author notes

  1. Bárbara B. Sousa and Marcos F. Q. Sousa contributed equally to this work.

Authors and Affiliations

  1. Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028, Lisboa, Portugal

    Bárbara B. Sousa, Marta C. Marques, João D. Seixas & Gonçalo J. L. Bernardes

  2. Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. Da República, EAN, 2780-157, Oeiras, Portugal

    Marcos F. Q. Sousa, José A. Brito, Pedro M. Matias & António Roldão

  3. Instituto de Biologia Experimental e Tecnológica, Avenida da República, Quinta Do Marquês, 2780-157, Oeiras, Portugal

    Marcos F. Q. Sousa, Pedro M. Matias & António Roldão

  4. Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK

    Gonçalo J. L. Bernardes

Authors
  1. Bárbara B. Sousa
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  2. Marcos F. Q. Sousa
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  3. Marta C. Marques
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  4. João D. Seixas
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  7. Gonçalo J. L. Bernardes
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  8. António Roldão
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Correspondence to António Roldão.

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Sousa, B.B., Sousa, M.F.Q., Marques, M.C. et al. A scalable insect cell-based production process of the human recombinant BMX for in-vitro covalent ligand high-throughput screening. Bioprocess Biosyst Eng 44, 209–215 (2021). https://doi.org/10.1007/s00449-020-02421-6

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