Adding ‘splice’ to protein engineering

Holford, Mandë; Muir, Tom W.

doi:10.1016/s0969-2126(98)00097-5

Cited by 14 publications

(7 citation statements)

References 49 publications

(61 reference statements)

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“…To derive quantitative data on the effect of serine phosphorylation on eIF4E interaction with the cap, we have generated, by the unique IPL technique (Holford et al 1998), an eIF4E protein that is specifically phosphorylated at Ser 209 and used it in a novel fluorometric time-synchronized titration method (Niedzwiecka et al 2002). The uncertainty of the determined K as values does not exceed 5%, except for the weak binding m 7 GMP (less than 10%).…”

Section: Discussionmentioning

confidence: 99%

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Phosphorylation of eIF4E attenuates its interaction with mRNA 5′ cap analogs by electrostatic repulsion: Intein-mediated protein ligation strategy to obtain phosphorylated protein

Zuberek¹,

Wysłouch‐Cieszyńska²,

Niedzwiecka³

et al. 2003

RNA

120

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Phosphorylation of the eukaryotic initiation factor eIF4E in response to mitogenic stimuli and cytokines is implicated in the regulation of the initiation step of translation. It still remains unclear how the phosphorylation of eIF4E regulates the translation. To address this problem, we applied a unique technique in protein engineering, intein-mediated protein ligation, to synthesize eIF4E, which is selectively phosphorylated at Ser 209. Using selectively chosen synthetic cap analogs, we compared quantitatively the cap affinity for phosphorylated and unphosphorylated eIF4E by a fluorometric time-synchronized titration method. A 1.5-to 4.5-fold reduction of the cap affinity for phosphorylated eIF4E was observed, depending on the negative charge of the 5-to-5 phosphate chains as well as the presence of a longer tetraribonucleotide strand. Possible implications for understanding the regulation of eIF4E functioning, cap complex formation, and stability, are discussed.

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Section: Discussionmentioning

confidence: 99%

“…A new technique in protein engineering, that is, inteinmediated protein ligation (IPL; Holford and Muir 1998;Xu and Evans 2001), has been applied to generate pure eIF4E, selectively phosphorylated at Ser 209 (Fig. 2).…”

Section: Introductionmentioning

confidence: 99%

Phosphorylation of eIF4E attenuates its interaction with mRNA 5′ cap analogs by electrostatic repulsion: Intein-mediated protein ligation strategy to obtain phosphorylated protein

Zuberek¹,

Wysłouch‐Cieszyńska²,

Niedzwiecka³

et al. 2003

RNA

120

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“…Intein‐mediated purification with an affinity chitin‐binding tag (the IMPACT system) utilizes the inducible self‐cleavage activity of a protein splicing element, that is, an intein 29, 30. The desired target protein is bacterially expressed as a fusion protein with a C‐terminal intein chitin‐binding domain (CBD) tag.…”

Section: Introductionmentioning

confidence: 99%

Cellular Internalization of Enhanced Green Fluorescent Protein Ligated to a Human Calcitonin-Based Carrier Peptide

et al. 2002

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“…For building up a stable peptide bond the protein and the peptide were linked by a native chemical ligation strategy [50]. Thus, the protein thioester was prepared via the IMPACT system (intein-mediated purification with an affinity chitin-binding tag) that utilizes the inducible self-cleavage activity of a protein splicing element, that is, an intein [51,52]. EGFP was cloned and expressed with a C-terminal intein-chitin binding domain (CBD) tag.…”

Section: Use Of Hct(9-32) and Its Derivative Hct(9-32)-br As Deliverymentioning

confidence: 99%

Calcitonin-Derived Carrier Peptides

Neundorf

Ag²

2005

CPD

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The recent discovery of carrier peptides offers new opportunities to translocate several bioactive molecules into the cytoplasm. Previous studies have shown that human calcitonin (hCT) and selected C-terminal sequences translocate in nasal epithelium. Moreover, the hCT(9-32) fragment was found to internalize efficiently a number of substances like fluorophores, nucleic acids or the enhanced green fluorescent protein (EGFP). In order to understand the uptake mechanism interactions of hCT(9-32) with membrane models of different lipid compositions have been investigated. From these studies it was possible to shed light on the conformational state of the peptide in the presence of membrane-like conditions. Further insight into the translocation mechanism was provided by fluorescence microscopy of truncated sequences of hCT that were shown to penetrate the plasma membrane and to distribute in a sectoral, punctuated pattern supporting an endocytotic internalization pathway as previously suggested.

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Adding ‘splice’ to protein engineering

Cited by 14 publications

References 49 publications

Phosphorylation of eIF4E attenuates its interaction with mRNA 5′ cap analogs by electrostatic repulsion: Intein-mediated protein ligation strategy to obtain phosphorylated protein

Phosphorylation of eIF4E attenuates its interaction with mRNA 5′ cap analogs by electrostatic repulsion: Intein-mediated protein ligation strategy to obtain phosphorylated protein

Cellular Internalization of Enhanced Green Fluorescent Protein Ligated to a Human Calcitonin-Based Carrier Peptide

Calcitonin-Derived Carrier Peptides

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