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 (His 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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http://dx.doi.org/10.1038/s41598-018-30435-4 | DOI Listing |
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Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, 28040 Madrid, Spain.
As part of the intestinal microbiota, can elicit a humoral response in the gastrointestinal tract (GIT) that is mainly directed toward hyphal antigens. This response has been implicated in controlling the invasive form of the fungus and maintaining the yeast as an innocuous commensal. However, the specific targets of this response are still unknown.
View Article and Find Full Text PDFACS Chem Neurosci
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Department of Occupational Health, School of Public Health, Shanxi Medical University, Taiyuan, Shanxi 030001, China.
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View Article and Find Full Text PDFCancer Immunol Res
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Vanderbilt University, Nashville, TN, United States.
Tumor-specific HLA class I expression is required for cytotoxic T-cell elimination of cancer cells expressing tumor-associated or neo-antigens. Cancers downregulate antigen presentation to avoid adaptive immunity. The highly polymorphic nature of the genes encoding these proteins, coupled with quaternary-structure changes after formalin fixation, complicate detection by immunohistochemistry.
View Article and Find Full Text PDFJ Med Chem
January 2025
Department of Pharmaceutical and Cell Biological Chemistry, Pharmaceutical Institute, University of Bonn institution, An der Immenburg 4, Bonn 53121, Germany.
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View Article and Find Full Text PDFCRISPR J
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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system has revolutionized targeted mutagenesis, but screening for mutations in large sample pools can be time-consuming and costly. We present an efficient and cost-effective polymerase chain reaction (PCR)-based strategy for identifying edited mutants in the T generation. Unlike previous methods, our approach addresses the challenges of large progeny populations by using T generation sequencing results for genotype prediction.
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