A scalable insect cell-based production process of the human recombinant BMX for in-vitro covalent ligand high-throughpu
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A scalable insect cell‑based production process of the human recombinant BMX for in‑vitro covalent ligand high‑throughput screening Bárbara B. Sousa1 · Marcos F. Q. Sousa2,3 · Marta C. Marques1 · João D. Seixas1 · José A. Brito2 · Pedro M. Matias2,3 · Gonçalo J. L. Bernardes1,4 · António Roldão2,3 Received: 24 February 2020 / Accepted: 31 July 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
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 twostep 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. Keywords hrBMX production · IC-BEVS · Bioprocess development · hrBMX crystallization · Cancer therapy
Introduction Bárbara B. Sousa and Marcos F. Q. Sousa contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00449-020-02421-6) contains supplementary material, which is available to authorized users. * António Roldão [email protected] 1
Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649‑028 Lisboa, Portugal
2
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. Da República, EAN, 2780‑157 Oeiras, Portugal
3
Instituto de Biologia Experimental e Tecnológica, Avenida da República, Quinta Do Marquês, 2780‑157 Oeiras, Portugal
4
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK
Bone Marrow Tyrosine kinase in the chromosome X (BMX) is a member of the TEC family of non-receptor kinases [1] and plays an important role in a variety of critical physiological and pathological processes, including tumorigenicity, cells motility, adhesion, angiogenesis, proliferation, and differentiation [2
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