An amalgamation of solid phase peptide synthesis and ribosomal peptide synthesis

Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, NY 10021, USA.
Biopolymers (Impact Factor: 2.39). 01/2008; 90(3):406-14. DOI: 10.1002/bip.20810
Source: PubMed

ABSTRACT Expressed protein ligation (EPL) is a protein semisynthesis technique that allows the site-specific introduction of unnatural amino acids and biophysical probes into proteins. In the present study, we illustrate the utility of the approach through the generation of two semisynthetic proteins bearing spectroscopic probes. Dihydrofolate reductase containing a single (13)C probe in an active site loop was generated through the ligation of a synthetic peptide-alpha-thioester to a recombinantly generated fragment containing an N-terminal Cys. Similarly, c-Crk-II was assembled by the sequential ligation of three recombinant polypeptide building blocks, allowing the incorporation of (15)N isotopes in the central domain of the protein. These examples showcase the scope of the protein ligation strategy for selective introduction of isotopic labels into proteins, and the protocols described will be of value to those interested in using EPL on other systems.

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    • "Sequential ligation of three recombinant polypeptides. The incorporation of isotopes in the central domain of the protein(Blaschke et al., 2000; Ottesen et al., 2008). "
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    ABSTRACT: Nuclear magnetic resonance (NMR) spectroscopy has emerged as one of the principle techniques of structural biology. It is not only a powerful method for elucidating the three-dimensional structures under near physiological conditions but also a convenient method for studying protein-ligand interactions and protein dynamics. A major drawback of macromolecular NMR is its size limitation, caused by slower tumbling rates and greater complexity of the spectra as size increases. Segmental isotopic labeling allows for specific segment(s) within a protein to be selectively examined by NMR, thus significantly reducing the spectral complexity for large proteins and allowing for the application of a variety of solution-based NMR strategies. Two related approaches are generally used in the segmental isotopic labeling of proteins: expressed protein ligation and protein trans-splicing. Here, we describe the methodology and recent application of expressed protein ligation and protein trans-splicing for NMR structural studies of proteins and protein complexes. We also describe the protocol used in our lab for the segmental isotopic labeling of a 50-kDa protein Csk (C-terminal Src kinase) using expressed protein ligation methods.
    Methods in enzymology 02/2009; 462:151-75. DOI:10.1016/S0076-6879(09)62008-5 · 2.09 Impact Factor
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    ABSTRACT: Histone post-translational modifications are essential for regulating and facilitating biological processes such as RNA transcription and DNA repair. Fifteen modifications are located in the DNA-histone dyad interface and include the acetylation of H3-K115 (H3-K115Ac) and H3-K122 (H3-K122Ac), but the functional consequences of these modifications are unknown. We have prepared semisynthetic histone H3 acetylated at Lys-115 and/or Lys-122 by expressed protein ligation and incorporated them into single nucleosomes. Competitive reconstitution analysis demonstrated that the acetylation of H3-K115 and H3-K122 reduces the free energy of histone octamer binding. Restriction enzyme kinetic analysis suggests that these histone modifications do not alter DNA accessibility near the sites of modification. However, acetylation of H3-K122 increases the rate of thermal repositioning. Remarkably, Lys --> Gln substitution mutations, which are used to mimic Lys acetylation, do not fully duplicate the effects of the H3-K115Ac or H3-K122Ac modifications. Our results are consistent with the conclusion that acetylation in the dyad interface reduces DNA-histone interaction(s), which may facilitate nucleosome repositioning and/or assembly/disassembly.
    Journal of Biological Chemistry 06/2009; 284(35):23312-21. DOI:10.1074/jbc.M109.003202 · 4.57 Impact Factor
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    ABSTRACT: A novel instrument was developed using a multi-wavelength pulsed LED array with in-column optic-fiber induced fluorescence detection by capillary electrophoresis. The light from 2 different wavelength LEDs (450 nm and 480 nm) was pulsed for short intervals at high intensity. The beam from each LED was collimated and reshaped with the gradient index (GRIN) lens group to achieve a highly effective coupling between LED light source and an optical fiber. The optical fiber was placed inside the capillary for in-capillary LED-induced fluorescence detection. The advantages of this system were validated by the simultaneous determination of vitamin B2 and fluorescein. Detection limits for vitamin B2 and fluorescein were estimated to be 5 nM and 0.29 nM (S/N=3), respectively. The relative standard deviations (RSDs, n=6) of the both compounds for migration time and peak area were better than 0.83%, 2.20% and 1.21%, 2.75%, respectively. The method was applied to the determination of vitamin B2 in commercial tablets and fluorescein in fluorescein sodium injection and the recoveries obtained were in the range of 96.6-102.0% and 99.9-102.8%, respectively. It was also applied to human serum, where the recoveries were found to be in the range of 94.4-97.0% and 92.6-96.4%, respectively. The system has been successfully applied in separation and determination of the both biological samples with acceptable analytical performance.
    Talanta 12/2010; 83(2):521-6. DOI:10.1016/j.talanta.2010.09.047 · 3.55 Impact Factor
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