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

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Richardson , D , Itkonen , J , Nievas , J , Urtti , A & Casteleijn , M G 2018 , ' Accelerated pharmaceutical protein development with integrated cell free expression, purification, and bioconjugation ' , Scientific Reports , vol. 8 , no. 1 , 11967 . https://doi.org/10.1038/s41598-018-30435-4

Title: Accelerated pharmaceutical protein development with integrated cell free expression, purification, and bioconjugation
Author: Richardson, Dominique; Itkonen, Jaakko; Nievas, Julia; Urtti, Arto; Casteleijn, Marco G.
Contributor: University of Helsinki, Faculty of Pharmacy
University of Helsinki, Faculty of Pharmacy
University of Helsinki, Drug Research Program
University of Helsinki, Faculty of Pharmacy
Date: 2018-08-10
Language: eng
Number of pages: 11
Belongs to series: Scientific Reports
ISSN: 2045-2322
URI: http://hdl.handle.net/10138/239090
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.
Subject: 317 Pharmacy
ESCHERICHIA-COLI
PHOTOLABILE LINKER
SPLIT INTEINS
SYSTEM
QUANTIFICATION
STREPTAVIDIN
OPTIMIZATION
STRATEGY
COST
GENE
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