Native Chemical Ligation of Polypeptides (original) (raw)
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Chemical Protein Synthesis by Native Chemical Ligation and Variations Thereof
Chinese Journal of Chemistry, 2019
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Chemical Protein Synthesis by Solid Phase Ligation of Unprotected Peptide Segments
Journal of the American Chemical Society, 1999
In this paper we describe "solid phase chemical ligation" (SPCL), the application of the principles of polymer-supported organic synthesis to the construction of large polypeptide chains for the total chemical synthesis of proteins. In this method, each building block used is an unprotected peptide segment of 20 or more amino acids. These are consecutively reacted by chemical ligation, the chemoselective reaction of the unprotected peptide segments from aqueous solution, to make the polymer-supported target polypeptide. In a final step, the assembled full-length target polypeptide is released from the aqueous-compatible polymer support.
Contemporary Methods for Peptide and Protein Synthesis
Current Organic Chemistry, 2001
This review describes current methods for peptide and protein syntheses, largely from a strategic point of view. The solid-phase method is useful for the rapid preparation of peptides. Two major synthetic strategies have been adopted by this method, namely, the Boc and Fmoc strategies. At the final stage of the Boc solidphase method, a protected peptide resin is treated with a strong acid to obtain a free peptide. On the other hand, in the Fmoc solid-phase method, a free peptide is obtained by treating a protected peptide resin with a weak acid. Both solid phase methods are quite useful for the preparation of peptides with molecular weights in the vicinity of five thousand. Ligation methods were developed to overcome the molecular weight barrier existing in a solid phase method. Building blocks used for ligation are prepared by the solid phase method, or more recently by biological methods. All the current ligation methods that produce a native peptide bond use peptide C-terminal thiocarboxylic acids or thioesters as building blocks. Blake et al. developed a selective activation method of the C-terminal carbonyl group by the combination of thiocarboxylic acid and silver ions. Based on this approach, a thioester method was developed, in which partially protected peptide thioesters are used as building blocks. Subsequently, a new ligation method was developed using peptide thioesters, in which protecting group is no longer necessary. The discovery of protein splicing phenomenon added a biological route to the preparation of peptide thioesters. A partially protected peptides segment can be also derived from an expressed peptide segment. Polypeptides with a molecular weight of more than 10 thousand can be routinely synthesized.
Biopolymers, 1999
Here we describe the results of studies designed to explore the scope and limitations of expressed protein ligation (EPL), a protein semisynthesis approach that allows unnatural amino acids to be site specifically introduced into large proteins. Using Src homology 3 domains from the proteins c-Abl and c-Crk as model systems, we show here that EPL can be performed in the presence of moderate concentrations of the chemical denaturant, guanidine hydrochloride, and the organic solvent dimethylsulfoxide. Use of these solubilizing agents allowed the successful preparation of two semisynthetic proteins, 10 and 12, both of which could not be prepared using standard procedures due to the low solubility of the synthetic peptide reactants in aqueous buffers. We also report the results of thiolysis and kinetic studies which indicate that stable alkyl thioester derivatives of recombinant proteins can be generated for storage and purification purposes, and that 2-mercaptoethanesulfonic acid compares favorably with thiophenol as the thiol cofactor for EPL reactions, while having superior handling properties. Finally, we describe the semisynthesis of the fluorescein/ rhodamine-containing construct (12) and the ketone-containing construct . The efficiency of these two syntheses indicates that EPL offers a facile way of incorporating these important types of biophysical and biochemical probes into proteins.
Total synthesis of cytochrome b562 by native chemical ligation using a removable auxiliary
Proceedings of the National Academy of Sciences, 2001
We have completed the total chemical synthesis of cytochrome b562 and an axial ligand analogue, [SeMet 7 ]cyt b562, by thioestermediated chemical ligation of unprotected peptide segments. A novel auxiliary-mediated native chemical ligation that enables peptide ligation to be applied to protein sequences lacking cysteine was used. A cleavable thiol-containing auxiliary group, 1-phenyl-2-mercaptoethyl, was added to the ␣-amino group of one peptide segment to facilitate amide bond-forming ligation. The amine-linked 1-phenyl-2-mercaptoethyl auxiliary was stable to anhydrous hydrogen fluoride used to cleave and deprotect peptides after solid-phase peptide synthesis. Following native chemical ligation with a thioester-containing segment, the auxiliary group was cleanly removed from the newly formed amide bond by treatment with anhydrous hydrogen fluoride, yielding a fulllength unmodified polypeptide product. The resulting polypeptide was reconstituted with heme and folded to form the functional protein molecule. Synthetic wild-type cyt b562 exhibited spectroscopic and electrochemical properties identical to the recombinant protein, whereas the engineered [SeMet 7 ]cyt b562 analogue protein was spectroscopically and functionally distinct, with a reduction potential shifted by Ϸ45 mV. The use of the 1-phenyl-2mercaptoethyl removable auxiliary reported here will greatly expand the applicability of total protein synthesis by native chemical ligation of unprotected peptide segments.
Chemical synthesis of proteins: a tool for protein labeling
2010
The need for protein modification strategies pag. Chemical synthesis of protein pag. Classical organic synthesis in solution pag. Solid phase peptide synthesis pag. Chemical ligation reaction pag. Chemical ligation of unprotected peptide segments pag. Chemical ligation reactions yielding a non-native link pag. Native Chemical Ligation pag. Expressed Protein Ligation pag. Performing Native Chemical Ligation and Expressed Protein Ligation pag. Production of thioester peptides pag. Production of N-terminal Cys peptide pag. Ligation of multiple peptide fragments pag. Protein semisynthesis by trans-splicing pag. Applications of NCL and EPL pag. Introduction of fluorescent probes pag. Introduction of isotopic probes pag. Introduction of post-translational modifications: phosphorylation, glycosylation, lipidation, ubiquitylation pag. EXPERIMENTAL SECTION pag. Materials and Instruments pag. Methods pag. Antibiotics pag. Solid and liquid media for bacterial strains pag. Preparation of E. coli TOP F'10 competent cells and transformation by electroporation pag. Preparation of E. coli competent cells and transformation by heat shock pag. Electrophoretic analysis of DNA pag. Electrophoretic analysis of proteins (SDS-PAGE) pag. Determination of the protein concentration pag. Bioinformatic tools pag. Cloning procedure pag. Purification pag. First labeling reaction pag. Native Chemical Ligation with L-Cys pag. Second labeling reaction pag. Mono-labeled and unlabeled control constructs preparation pag. Spectroscopic characterization of CTPR3 protein variants pag. CD characterization pag. Fluorescence anisotropy measurements pag. Ensemble-FRET studies on Doubly-labeled CTPR3 protein variants pag. Chemical denaturation studies by CD and FRET pag. CONCLUSION pag. ABBREVIATIONS Acm acetamidomethyl AW azatryptophan BAL backbone amide linker Boc t-butoxycarbonyl BSA bovine serum albumin c-Abl Abelson nonreceptor protein tyrosine kinase CD circular dicroism ctpr3 Consensus Tetratrico Peptide Repeat protein 3 gene CTPR3 Consensus Tetratrico Peptide Repeat protein 3 Dab dabcyl Dansyl 5-(dimethylamino)-naphtalene-sulfonamide DBU 1,8-diazabicyclo[5.4.0]undec-7-ene Dbz diaminobenzoic acid DCM dichloromethane DIPEA N,N-diisopropylethylamine DMAP 4-Dimethylaminopyridine DMF dimethylformamide DMSO dimethylsulfoxide DNA deoxyribonucleic acid dNTP deoxy-nucleotide tri-phosphate ε extinction coefficient E FRET efficiency E. coli Escherichia coli EDT ethandithiol EDTA ethylene-diamino-tetraacetic acid EPL Expressed Protein Ligation ESI electron spray ionization source FCS Fluorescence Correlation Spectroscopy Fmoc fluorenylmethyloxycarbonyl chloride FP Fluorescence Polarization FPLC Fast Protein Liquid Chromatography FRET Förster Resonance Energy Transfer GdnHCl guanidinium hydrochloride GFP Green Fluorescent Protein Gla PDA photo diode array pI isoelectric point
Protein Synthesis by Solid-Phase Chemical Ligation Using a Safety Catch Linker
The Journal of Organic Chemistry, 2000
The native chemical ligation reaction has been used extensively for the synthesis of the large polypeptides that correspond to folded proteins and domains. The efficiency of the synthesis of the target protein is highly dependent on the number of peptide segments in the synthesis. Assembly of proteins from multiple components requires repeated purification and lyophilization steps that give rise to considerable handling losses. In principle, performing the ligation reactions on a solid support would eliminate these inefficient steps and increase the yield of the protein assembly. A new strategy is described for the assembly of large polypeptides on a solid support that utilizes a highly stable safety catch acid-labile linker. This amide generating linker is compatible with a wide range of N-terminal protecting groups and ligation chemistries. The utility of the methodology is demonstrated by a three-segment synthesis of vMIP I, a chemokine that contains all 20 natural amino acids and has two disulfide bonds. The crude polypeptide product was recovered quantitatively from the solid support and purified in 20%-recovered yield. This strategy should facilitate the synthesis of large polypeptides and should find useful applications in the assembly of protein libraries.
Semisynthesis of Dimeric Proteins by Expressed Protein Ligation
Organic Letters, 2008
General experimental All chemicals and solvents are commercially available (Novabiochem, Sigma-Aldrich, LabScan) and were used without further purification. The pTXB1 vector and the chitin resin were purchased at New England Biolabs; pTrcHisA and Ni-NTA resin were from Invitrogen. Column chromatography was performed on Fluka silica gel 60 (size: 0.04-0.063mm). 1 Hand 13 C-NMR spectra were recorded on a Varian Innova instrument (600 MHz) at room temperature. All chemical shifts are expressed in ppm with respect to the signals of the residual protonated solvents (CDCl 3 or DMSO d 6). LCMS analyses of the proteins were run on a Thermo Finnigan instrument equipped with a LCQ Deca XPMax ES source using a Phenomenex Jupiter 5μ C4 300Ǻ, 250x2.00 mm column. LCMS analyses of the linker were run on a Thermo Finnigan instrument equipped with a MSQ ES source using a Phenomenex Jupiter 5μ C18 300Ǻ, 150x4.6 mm column. Preparative HPLC was performed on a Phenomex Jupiter 10μ Proteo 90Ǻ 250x10.00 mm column. Synthesis of the linker N-N' bis-cysteinyl-ethylendiamine To a solution of ethylendiamine (30.1μL, 0.45mmol) and DIPEA (485.5 μL, 2.7 mmol) in dry DCM (300 μL) was added a solution of BocCys(Trt)OH (500mg, 1.08 mmol), PyBOP (467 mg, 0.90 mmol) and HOBT (121.4 mg, 0.90 mmol) in dry DCM (2 mL). The reaction was stirred overnight at room temperature. The mixture was extracted with a 5% NaHCO 3 aqueous solution. The organic phase was dried over MgSO 4 and the solvent was evaporated under reduced pressure. The residue was purified by silica gel flash chromatography (ethyl