Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

Heil, Christina S.; Rittner, Alexander; Goebel, Bjarne; Beyer, Dániel; Grininger, Martin

doi:10.1038/s41598-018-33115-5

Cited by 12 publications

(15 citation statements)

References 52 publications

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“…Besides the spectroscopic characterization, we tested the general biofunctionality of the nanosheets. To this end, SPR measurements (see SI Figure S5) were employed using bovine serum albumin (BSA, without His-tag to test for unspecific bioadhesion, 67 kDa) and His 8 -tagged green fluorescent protein (His 8 -GFP, as a specific analyte, 39 kDa) . As shown in Figure b, after incubation of the nanosheet with Ni 2+ , biorecognition sites with a strong affinity toward His-tagged biomolecules together with a biorepellent background due to the PG functionalization are provided.…”

Section: Results and Discussionmentioning

confidence: 99%

Smart Molecular Nanosheets for Advanced Preparation of Biological Samples in Electron Cryo-Microscopy

et al. 2020

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Transmission electron cryo-microscopy (cryoEM) of vitrified biological specimens is a powerful tool for structural biology. Current preparation of vitrified biological samples starts off with sample isolation and purification, followed by the fixation in a freestanding layer of amorphous ice. Here, we demonstrate that ultrathin (∼10 nm) smart molecular nanosheets having specific biorecognition sites embedded in a biorepulsive layer covalently bound to a mechanically stable carbon nanomembrane allow for a much simpler isolation and structural analysis. We characterize in detail the engineering of these nanosheets and their biorecognition properties employing complementary methods such as X-ray photoelectron and infrared spectroscopy, atomic force microscopy as well as surface plasmon resonance measurements. The desired functionality of the developed nanosheets is demonstrated by in situ selection of a His-tagged protein from a mixture and its subsequent structural analysis by cryoEM.

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Section: Results and Discussionmentioning

confidence: 99%

Smart Molecular Nanosheets for Advanced Preparation of Biological Samples in Electron Cryo-Microscopy

et al. 2020

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“…This finding hints at less efficient suppression of the in-frame amber codon in the Z HER2:2891 K50am sequence context, and an alternative site for ncAA incorporation could increase the expression level of Z HER2:2891 K50AzK. 68 Many ncAAs, and notably those carrying reactive handles such as AzK, are rather expensive, e.g., AzK comes at a price of 112−200 €/g. 69 While this appears tolerable for shake flask cultures, the price of the ncAA is critical for large-scale high cell density bioreactor cultures, and it is mandatory to use "as much as required but as little as possible".…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Decoupling Protein Production from Cell Growth Enhances the Site-Specific Incorporation of Noncanonical Amino Acids in E. coli

Casas

Stargardt²,

Mairhofer³

et al. 2020

ACS Synth. Biol.

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The site-specific incorporation of noncanonical amino acids (ncAAs) into proteins by amber stop codon suppression has become a routine method in academic laboratories. This approach requires an amber suppressor tRNACUA to read the amber codon and an aminoacyl-tRNA synthetase to charge the tRNACUA with the ncAA. However, a major drawback is the low yield of the mutant protein in comparison to the wild type. This effect primarily results from the competition of release factor 1 with the charged suppressor tRNACUA for the amber codon at the A-site of the ribosome. A number of laboratories have attempted to improve the incorporation efficiency of ncAAs with moderate results. We aimed at increasing the efficiency to produce high yields of ncAA-functionalized proteins in a scalable setting for industrial application. To do this, we inserted an ncAA into the enhanced green fluorescent protein and an antibody mimetic molecule using an industrial E. coli strain, which produces recombinant proteins independent of cell growth. The controlled decoupling of recombinant protein production from cell growth considerably increased the incorporation of the ncAA, producing substantially higher protein yields versus the reference E. coli strain BL21(DE3). The target proteins were expressed at high levels, and the ncAA was efficiently incorporated with excellent fidelity while the protein function was preserved.

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“…Sfp used for phosphopantetheinylation was produced in E. coli (Strain M15; helper plasmid pREP4 and pQE-60 encoding Sfp) and purified by His-tag affinity chromatography, dialysis overnight against imidazole free buffer (250 mM NaCl, 2 mM MgCl 2 , 10% glycerol, 50 mM HEPES (pH 8.0 adjusted with HCl)) and SEC (HiLoad Superdex 200 16/60 pg) 56 . Holo mm ACP was produced in E. coli and purified by His-tag affinity chromatography (basic buffer: 200 mM NaCl, 35 mM K 2 HPO 4 and 15 mM KH 2 PO 4 (pH 7.4, adjusted with NaOH or HCl), 10% glycerol; washing buffer contained 20 mM imidazole and elution buffer contained 300 mM imidazole) and SEC (HiLoad Superdex 75 16/60 pg), similar as described for ACP-GFP by Heil and Rittner et al 57 Pooled SEC fractions of Sfp and mm ACP were aliquoted, frozen in liquid nitrogen and stored at − 80 °C. The modification of mm ACP and mt Dod- mm ACP (both variants with H8-Tag and without) with CoA 488 (CoA modified with ATTO-TEC dye ATTO 488, NEB #S9348) by Sfp was conducted in phosphate borate buffer (pH 7.4, adjusted with HCl) at 25 °C for 1 h at following conditions: 10 µM mt Dod- mm ACP or mm ACP, 4 µM Sfp, 1 mM DTT, 10 mM MgCl 2 and 10 µM CoA 488.…”

Section: Methodsmentioning

confidence: 99%

Dodecin as carrier protein for immunizations and bioengineering applications

Bourdeaux

Kopp

Lautenschläger

et al. 2020

Sci Rep

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In bioengineering, scaffold proteins have been increasingly used to recruit molecules to parts of a cell, or to enhance the efficacy of biosynthetic or signalling pathways. For example, scaffolds can be used to make weak or non-immunogenic small molecules immunogenic by attaching them to the scaffold, in this role called carrier. Here, we present the dodecin from Mycobacterium tuberculosis (mtDod) as a new scaffold protein. MtDod is a homododecameric complex of spherical shape, high stability and robust assembly, which allows the attachment of cargo at its surface. We show that mtDod, either directly loaded with cargo or equipped with domains for non-covalent and covalent loading of cargo, can be produced recombinantly in high quantity and quality in Escherichia coli. fusions of mtDod with proteins of up to four times the size of mtDod, e.g. with monomeric superfolder green fluorescent protein creating a 437 kDa large dodecamer, were successfully purified, showing mtDod's ability to function as recruitment hub. further, mtDod equipped with SYNZIP and SpyCatcher domains for post-translational recruitment of cargo was prepared of which the mtDod/Spycatcher system proved to be particularly useful. In a case study, we finally show that mtDod-peptide fusions allow producing antibodies against human heat shock proteins and the C-terminus of heat shock cognate 70 interacting protein (cHip).

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Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

Cited by 12 publications

References 52 publications

Smart Molecular Nanosheets for Advanced Preparation of Biological Samples in Electron Cryo-Microscopy

Smart Molecular Nanosheets for Advanced Preparation of Biological Samples in Electron Cryo-Microscopy

Decoupling Protein Production from Cell Growth Enhances the Site-Specific Incorporation of Noncanonical Amino Acids in E. coli

Dodecin as carrier protein for immunizations and bioengineering applications

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