Abstract
The use of living cells for the synthesis of pharmaceutical proteins, though state-of-the-art, is hindered by its lengthy process comprising of many steps that may affect the protein’s stability and activity. We aimed to integrate protein expression, purification, and bioconjugation in small volumes coupled with cell free protein synthesis for the target protein, ciliary neurotrophic factor. Split-intein mediated capture by use of capture peptides onto a solid surface was efficient at 89–93%. Proof-of-principle of light triggered release was compared to affinity chromatography (His6 fusion tag coupled with Ni-NTA). The latter was more efficient, but more time consuming. Light triggered release was clearly demonstrated. Moreover, we transferred biotin from the capture peptide to the target protein without further purification steps. Finally, the target protein was released in a buffer-volume and composition of our choice, omitting the need for protein concentration or changing the buffer. Split-intein mediated capture, protein trans splicing followed by light triggered release, and bioconjugation for proteins synthesized in cell free systems might be performed in an integrated workflow resulting in the fast production of the target protein.
Highlights
In traditional biotechnology, cells are utilized for a variety of different processes[1], and more recently during the eras of genetics and molecular biology, one important process is recombinant protein production[2,3,4,5]
We demonstrate a workflow for the synthesis, purification, bioconjugation, and formulation of a pharmaceutical protein: human ciliary neurotrophic factor
Several cell-free systems were evaluated: E. coli BL21(DE3) S12 and S30 lysates (Figs S1 and S2), tobacco plant BY-2 cell lysates (BYL)[17], commercial kits based on E. coli (Promega, USA; Figs S1 and S2), wheat germ (Biotechrabbit, Germany; Fig. S3), and mammalian HeLa cells (ThermoFisher scientific, USA)
Summary
Cells are utilized for a variety of different processes[1], and more recently during the eras of genetics and molecular biology, one important process is recombinant protein production[2,3,4,5]. Cell free protein synthesis (CFPS) has shown its importance for pharmaceutical protein production, including the industrial production of a commercial protein drug (rhGM-CSF)[15] This product was still purified using traditional methods, such as weak anion exchange chromatography, followed by filtration and several size exclusion chromatography steps, with all their problems as stated before. Our approach utilizes cell free protein synthesis, split-intein mediated capture, light triggered release, moiety transfer upon release to facilitate bioconjugation, and formulating protein in the correct buffer at the desired protein concentration (see Fig. 1) This workflow omits many time-consuming steps of downstream protein production (e.g. the use of living cells, chromatography, dilution and re-concentrating, buffer exchange, purification of the bioconjugates, and re-folding). The proof-of-principle for this method is demonstrated here using ciliary neurotrophic factor (CNTF) and green fluorescent protein (GFP)
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