Accelerated pharmaceutical protein development with integrated cell free expression, purification, and bioconjugation

Dominique Richardson, Jaakko Itkonen, Julia Nievas, Arto Urtti, Marco G. Casteleijn (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

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.
Original languageEnglish
Article number11967
Pages (from-to)2045-2322
JournalScientific Reports
Volume8
Issue number1
DOIs
Publication statusPublished - 10 Aug 2018
MoE publication typeA1 Journal article-refereed

Fingerprint

Pharmaceutical Preparations
Proteins
Inteins
His-His-His-His-His-His
Light
Buffers
Trans-Splicing
Protein Splicing
Ciliary Neurotrophic Factor
Peptides
Cell-Free System
Workflow
Protein Stability
Biotin
Affinity Chromatography

Keywords

  • proteins
  • expression systems

Cite this

Richardson, Dominique ; Itkonen, Jaakko ; Nievas, Julia ; Urtti, Arto ; Casteleijn, Marco G. / Accelerated pharmaceutical protein development with integrated cell free expression, purification, and bioconjugation. In: Scientific Reports. 2018 ; Vol. 8, No. 1. pp. 2045-2322.
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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.",
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Accelerated pharmaceutical protein development with integrated cell free expression, purification, and bioconjugation. / Richardson, Dominique; Itkonen, Jaakko; Nievas, Julia; Urtti, Arto; Casteleijn, Marco G. (Corresponding Author).

In: Scientific Reports, Vol. 8, No. 1, 11967, 10.08.2018, p. 2045-2322.

Research output: Contribution to journalArticleScientificpeer-review

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