Multiple‐Site Labeling of Proteins with Unnatural Amino Acids

Loscha, Karin V.; Herlt, Anthony J.; Qi, Ruhu; Huber, Thomas; Ozawa, Kiyoshi; Otting, Gottfried

doi:10.1002/anie.201108275

Cited by 97 publications

(105 citation statements)

References 41 publications

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“…These methods require the introduction of spin-labels (Tamm et al 2007;Schiemann and Prisner 2007;Keizers and Ubbink 2011;Yagi et al 2011;Loscha et al 2012;Russo et al 2013) attached to or within a protein. In NMR-spectroscopic studies, attachment of a single paramagnetic lanthanide center (Keizers and Ubbink 2011) with an anisotropic Dv-tensor (Bertini et al 2002) has been employed to obtain PCSs, which report on the distance and radial coordinates of a nuclear spin with respect to the paramagnetic center.…”

Section: Introductionmentioning

confidence: 99%

Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy

et al. 2015

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We recently engineered encodable lanthanide binding tags (LBTs) into proteins and demonstrated their applicability in Nuclear Magnetic Resonance (NMR) spectroscopy, X-ray crystallography and luminescence studies. Here, we engineered two-loop-LBTs into the model protein interleukin-1b (IL1b) and measured 1 H, 15 Npseudocontact shifts (PCSs) by NMR spectroscopy. We determined the Dv-tensors associated with each Tm 3? -loaded loop-LBT and show that the experimental PCSs yield structural information at the interface between the two metal ion centers at atomic resolution. Such information is very valuable for the determination of the sites of interfaces in protein-protein-complexes. Combining the experimental PCSs of the two-loop-LBT construct IL1b-S2R2 and the respective single-loop-LBT constructs IL1b-S2, IL1b-R2 we additionally determined the distance between the metal ion centers. Further, we explore the use of two-loop LBTs loaded with Gd 3? as a novel tool for distance determination by Electron Paramagnetic Resonance spectroscopy and show the NMR-derived distances to be remarkably consistent with distances derived from Pulsed Electron-Electron Dipolar Resonance.

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Section: Introductionmentioning

confidence: 99%

Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy

et al. 2015

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“…Initially RNA ligase acylation methods were used [42][43][44][45][46][47][48][49][50][51][52][53][54] but as with other chemical ligation methods, were not usefully efficient, however, more recent developments are more promising [43]. The challenge remained to achieve high efficiency of orthogonal tRNA recharging with the unnatural amino acid, and not any of the natural amino acids, in situ during the course of the cell free reaction to generate high yields of homogeneous protein.…”

Section: In Vitro Expression Of Antibodies Containing Unnatural Aminomentioning

confidence: 99%

“…Removal of RF1, the release factor responsible for causing truncation at the amber stop codon, is complicated by the fact that deletion of the gene from cells is lethal in both microbial and eukaryotic systems. In cell-based systems there are efforts to generate RF1 deleted cell strains where RF2 is engineered to largely compensate for the lack of RF1 with efficient unnatural amino acid incorporation [48,49].…”

Section: Unnatural Amino Acid Incorporation Efficiencymentioning

confidence: 99%

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Unnatural Amino Acids in Novel Antibody Conjugates

Hallam

Smider

2014

Future Med. Chem.

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Antibody-drug conjugates are an important and emerging drug class for the treatment of cancer. Recent evidence strongly suggests that site-specific drug conjugation results in a homogenous population of molecules with more favorable activity and pharmacokinetic properties than randomly conjugated antibodies. Unnatural amino acids (uAAs) can be incorporated in recombinant proteins to enable unique orthogonal chemistries in comparison to the side chains of the natural 20 amino acids. Thus, uAAs present a novel platform for which to create next-generation antibody-drug conjugates. Furthermore, site-specific conjugation through uAAs can also enpower unique small molecule, bispecific, multispecific and other conjugates that could be important constructs for therapeutics, diagnostics and research reagents. Here, we review the progress in uAA incorporation and conjugate construction through both cell-based and -free approaches.

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“…This approach enabled the synthesis of decorated virus-like particles that could function as potential vaccines and imaging agents. CFPS systems have also been used to site-specifically incorporate ngeAAs at multiples sites in a protein without having to modify the source strain [26] or selectively removing a tagged release factor-1 (RF-1) to remove competition for the amber codon [27]. Moving forward, recent advances to improve ngeAA incorporation efficiency in vivo , such as deletion of RF-1 [27–29] or reassigning natural codons to expand the number of co-opted codons [30], should advance in vitro efforts that use crude extracts.…”

Section: Using Cell-free Synthetic Biology For Synthetic Chemistrymentioning

confidence: 99%

Cell-free biology: exploiting the interface between synthetic biology and synthetic chemistry

Harris

Jewett

2012

Current Opinion in Biotechnology

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Just as synthetic organic chemistry once revolutionized the ability of chemists to build molecules (including those that did not exist in nature) following a basic set of design rules, cell-free synthetic biology is beginning to provide an improved toolbox and faster process for not only harnessing but also expanding the chemistry of life. At the interface between chemistry and biology, research in cell-free synthetic systems is proceeding in two different directions: using synthetic biology for synthetic chemistry and using synthetic chemistry to reprogram or mimic biology. In the coming years, the impact of advances inspired by these approaches will make possible the synthesis of non-biological polymers having new backbone compositions, new chemical properties, new structures, and new functions.

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Multiple‐Site Labeling of Proteins with Unnatural Amino Acids

Cited by 97 publications

References 41 publications

Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy

Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy

Unnatural Amino Acids in Novel Antibody Conjugates

Cell-free biology: exploiting the interface between synthetic biology and synthetic chemistry

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