Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
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SHR Neuro Krebs Kardio Lipid Stoffw Microb
Stadler, KA; Becker, W; Darnhofer, B; Birner-Gruenberger, R; Zangger, K.
Overexpression of recombinant proteins containing non-canonical amino acids in Vibrio natriegens: p-azido-L-phenylalanine as coupling site for 19F-tags.
Amino Acids. 2022; 54(7):1041-1053
Doi: 10.1007/s00726-022-03148-2
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- Co-Autor*innen der Med Uni Graz
- Birner-Grünberger Ruth
- Darnhofer Barbara
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- Abstract:
- Vibrio natriegens is the fastest growing organism identified so far. The minimum doubling time of only 9.4 min, the ability to utilize over 60 different carbon sources and its non-pathogenic properties make it an interesting alternative to E. coli as a new production host for recombinant proteins. We investigated the ability of the engineered V. natriegens strain, Vmax™ Express, to incorporate the non-canonical amino acid (ncAA) p-azido-L-phenylalanine (AzF) into recombinant proteins for NMR applications. AzF was incorporated into enhanced yellow fluorescent protein (EYFP) and MlaC, an intermembrane transport protein, by stop codon suppression. AzF incorporation into EYFP resulted in an improved suppression efficiency (SE) of up to 35.5 ± 0.8% and a protein titer of 26.7 ± 0.7 mg/L. The expression levels of MlaC-AzF even exceeded those of E. coli BL21 cells. For the recording of 1H-15N and 19F NMR spectra, EYFP-AzF was expressed and isotopically labeled in minimal medium and the newly introduced azido-group was used as coupling site for NMR sensitive 19F-tags. Our findings show that Vmax is a flexible expression host, suitable for the incorporation of ncAAs in recombinant proteins with the potential to surpass protein yields of E. coli. The presented method suggests the implementation of V. natriegens for expression of isotopically labeled proteins containing ncAAs, which can be chemically modified for the application in protein-observed 19F-NMR.
- Find related publications in this database (using NLM MeSH Indexing)
- Amino Acids - chemistry
- Amino Acyl-tRNA Synthetases - metabolism
- Escherichia coli - genetics, metabolism
- Phenylalanine - metabolism
- Recombinant Proteins - genetics, metabolism
- Vibrio - administration & dosage
- Find related publications in this database (Keywords)
- Unnatural amino acids
- Orthogonal tRNA
- tRNA synthetase
- Stop codon suppression
- F-19-NMR