ORCID Profile
0000-0002-4511-0698
Current Organisations
Graz University of Technology
,
University of Sydney
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Publisher: Cold Spring Harbor Laboratory
Date: 20-12-2022
Abstract: RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use R NA:RN A b inding by S HAPE (RABS) to investigate microRNA-34a ( miR-34a ) binding its mRNA target, the silent information regulator 1 (m SIRT1 ), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.
Publisher: Springer International Publishing
Date: 2018
Publisher: Wiley
Date: 11-04-2018
Publisher: American Chemical Society (ACS)
Date: 14-06-2019
DOI: 10.1021/ACS.BIOCHEM.9B00403
Abstract: Isotopic labeling of recombinant proteins is crucial for studying proteins by liquid state NMR spectroscopy. Nowadays, conventional E. coli-based expression systems like BL21 (DE3) are typically used to express recombinant proteins. Still, the production of isotopically labeled proteins is often costly and time-consuming, and yields are not sufficient enough for structural studies. Here, we present Vibrio natriegens (Vmax) as an alternative expression system in M9 minimal medium. Due to our optimized M9 minimal medium and conditions and the early time point of induction, we obtained a 2- to 4-fold higher protein yield for two test proteins, FKBP and EYFP, compared to E. coli BL21 (DE3). Production of proteins in V. natriegens in minimal medium is not only more cost-effective and convenient but also less time-consuming than in E. coli. Comparing
Publisher: Elsevier BV
Date: 10-2021
Publisher: Wiley
Date: 16-02-2018
Publisher: Springer Science and Business Media LLC
Date: 07-01-2019
Publisher: Springer Science and Business Media LLC
Date: 13-04-2022
DOI: 10.1007/S00726-022-03148-2
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 1 H- 15 N and 19 F 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 19 F-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 19 F-NMR.
Publisher: Royal Society of Chemistry (RSC)
Date: 2017
DOI: 10.1039/C7CC06155B
Abstract: Trivalent pnictogens (Pn) enable the selective self-assembly between an engineered di-cysteine motif in a protein and a thiol-containing lanthanide (Ln) probe.
Publisher: Springer Science and Business Media LLC
Date: 21-03-2018
No related grants have been discovered for Walter Becker.