Site-Specific <sup>18</sup>F-Labeling of the Protein Hormone Leptin Using a General Two-Step Ligation Procedure

Flavell, Robert R.; Kothari, Paresh; Bar-Dagan, Maya; Synan, Michael; Vallabhajosula, Shankar; Friedman, Jeffrey M.; Muir, Tom W.; Ceccarini, Giovanni

doi:10.1021/ja801666z

Cited by 65 publications

(86 citation statements)

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“…Intein applications follow from the impressive ease with which these protein catalysts splice or cleave from some nonnative exteins (19,20). Recently devel- oped intein-based technologies include the fabrication of PET imaging agents (21) and protein chips, as well as the preparation of mixed biomacromolecules (20). An awareness of extein effects identified here will be useful not only for guiding optimal insertion sites in heterologous protein sequences, but also for using such effects for the control of inteins.…”

Section: Discussionmentioning

confidence: 91%

Modulation of intein activity by its neighboring extein substrates

Amitai

Callahan

Stanger

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

105

131

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Inteins comprise a large family of phylogenetically widespread self-splicing protein catalysts that colonize diverse host proteins. The evolutionary and functional relationship between the intein and the split-host protein, the exteins, is largely unknown. To probe an association, we developed an in vivo and in vitro intein assay based on FRET. The FRET assay reports cleavage of the intein from its N-terminal extein. Applying this assay to randomized extein libraries, we show that the nature of the extein substrate bordering the intein can profoundly influence intein activity. Residues proximal to the intein-splicing junction in both N-and C-terminal exteins can accelerate the N-terminal cleavage rate by >4-fold or attenuate cleavage by 1,000-fold, both resulting in compromised self-splicing efficiency. The existence and the magnitude of extein effects require consideration for maximizing the utility of inteins in biotechnological applications, and they predict biases in intein integration sites in nature.biotechnological application ͉ extein effects ͉ FRET assay for intein function ͉ intein spread A n intein separates 2 segments of a protein before catalyzing its self-excision and simultaneously reuniting the host protein components, or exteins. Many inteins are invasive elements at the DNA level, their exteins are diverse, and they occur in all 3 domains of life (1, 2). During protein splicing, the amino-and carboxy-terminal-f lanking exteins (N-and C-exteins) are thought of mainly as bystanders, with the exception of the first C-extein residue, which is invariably Cys, Ser, or Thr. Minimizing the significance of exteins in catalysis may seem reasonable from a chemical perspective, given their role as substrates. From a biological perspective also, the relationship of the extein to its intein is assumed to be less than mutualistic, in accord with a host-parasite interaction (1-3). However, the assumption of extein-independence is challenged by studies on 4 different inteins, demonstrating an influence of the adjacent N-extein (N-1) or 2 adjacent C-extein residues (Cϩ1, Cϩ2) on intein activity (4-8). Extein effects, potentially related to their folding status, were also apparent during attempts to interrupt the erythropoietin protein with a RecA intein (9). Furthermore, our analysis of an extein sequence database of Ͼ200 inteins (http:// bioinformatics.weizmann.ac.il/ϳpietro/inteins) suggests that bias exists for the N-1, N-2, Cϩ2, and Cϩ3 residues (Fig. S1 A).In the present work, we tested the limits of extein tolerance. We studied extein influence beyond a single position by randomization of either 2 or 4 extein residues nearest to the 2 intein junctions, with mutant libraries of 400-or 160,000-aa combinations, respectively. Our high-throughput approach, which offers considerable advantages over typical gel-based intein activity assays, employs FRET. Here, cyan and yellow fluorescent proteins (CFP and YFP) (10) act as FRET-active surrogate Nand C-exteins, respectively ( Fig. 1 A and B). They are joined by a f...

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Section: Discussionmentioning

confidence: 91%

Modulation of intein activity by its neighboring extein substrates

Amitai

Callahan

Stanger

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

105

131

View full text Add to dashboard Cite

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“…18 (19). We decided to test this radiochemistry for Z HER2:2891 in combination with the recently discovered aniline catalysis (20)(21)(22). With regard to the automation potential of the radiolabeling protocol, we also focused our attention on 2 simple 1-step methods.…”

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confidence: 99%

Three Methods for ¹⁸F Labeling of the HER2-Binding Affibody Molecule Z_HER2:2891 Including Preclinical Assessment

et al. 2013

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“…This precursor was used to incorporate an aminooxy functional group into the C terminus of leptin in an aniline-accelerated radiochemical oximation reaction, allowing for unambiguous bioactivity determination [65]. Further site-specific labeling has been obtained by using a silicon-fluoride acceptor (SiFA) reagent such as [ 18 F]SiFA-SH.…”

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confidence: 99%

Radiolabeling Strategies for Tumor-Targeting Proteinaceous Drugs

et al. 2014

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Owing to their large size proteinaceous drugs offer higher operative information content compared to the small molecules that correspond to the traditional understanding of druglikeness. As a consequence these drugs allow developing patient-specific therapies that provide the means to go beyond the possibilities of current drug therapy. However, the efficacy of these strategies, in particular "personalized medicine", depends on precise information about individual target expression rates. Molecular imaging combines non-invasive imaging methods with tools of molecular and cellular biology and thus bridges current knowledge to the clinical use. Moreover, nuclear medicine techniques provide therapeutic applications with tracers that behave like the diagnostic tracer. The advantages of radioiodination, still the most versatile radiolabeling strategy, and other labeled compounds comprising covalently attached radioisotopes are compared to the use of chelator-protein conjugates that are complexed with metallic radioisotopes. With the techniques using radioactive isotopes as a reporting unit or even the therapeutic principle, care has to be taken to avoid cleavage of the radionuclide from the protein it is linked to. The tracers used in molecular imaging require labeling techniques that provide site specific conjugation and metabolic stability. Appropriate choice of the radionuclide allows tailoring the properties of the labeled protein to the application required. Until the event of positron emission tomography the spectrum of nuclides used to visualize cellular and biochemical processes was largely restricted to iodine isotopes and 99m-technetium. Today, several nuclides such as 18-fluorine, 68-gallium and 86-yttrium have fundamentally extended the possibilities of tracer design and in turn caused the need for the development of chemical methods for their conjugation.

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Site-Specific ¹⁸F-Labeling of the Protein Hormone Leptin Using a General Two-Step Ligation Procedure

Cited by 65 publications

References 50 publications

Modulation of intein activity by its neighboring extein substrates

Modulation of intein activity by its neighboring extein substrates

Three Methods for ¹⁸F Labeling of the HER2-Binding Affibody Molecule Z_HER2:2891 Including Preclinical Assessment

Radiolabeling Strategies for Tumor-Targeting Proteinaceous Drugs

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Site-Specific 18F-Labeling of the Protein Hormone Leptin Using a General Two-Step Ligation Procedure

Cited by 65 publications

References 50 publications

Modulation of intein activity by its neighboring extein substrates

Modulation of intein activity by its neighboring extein substrates

Three Methods for 18F Labeling of the HER2-Binding Affibody Molecule ZHER2:2891 Including Preclinical Assessment

Radiolabeling Strategies for Tumor-Targeting Proteinaceous Drugs

Contact Info

Product

Resources

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Site-Specific ¹⁸F-Labeling of the Protein Hormone Leptin Using a General Two-Step Ligation Procedure

Three Methods for ¹⁸F Labeling of the HER2-Binding Affibody Molecule Z_HER2:2891 Including Preclinical Assessment