共查询到20条相似文献,搜索用时 31 毫秒
1.
Sakamoto I Tezuka K Fukae K Ishii K Taduru K Maeda M Ouchi M Yoshida K Nambu Y Igarashi J Hayashi N Tsuji T Kajihara Y 《Journal of the American Chemical Society》2012,134(12):5428-5431
Chemical synthesis of homogeneous human glycoproteins exhibiting bioactivity in vivo has been a challenging task. In an effort to overcome this long-standing problem, we selected interferon-β and examined its synthesis. The 166 residue polypeptide chain of interferon-β was prepared by covalent condensation of two synthetic peptide segments and a glycosylated synthetic peptide bearing a complex-type glycan of biological origin. The peptides were covalently condensed by native chemical ligation. Selective desulfurization followed by deprotection of the two Cys(Acm) residues gave the target full-length polypeptide chain of interferon-β bearing either a complex-type sialyl biantennary oligosaccharide or its asialo form. Subsequent folding with concomitant formation of the native disulfide bond afforded correctly folded homogeneous glycosyl-interferon-β. The chemically synthesized sialyl interferon-β exhibited potent antitumor activity in vivo. 相似文献
2.
Pentelute BL Gates ZP Dashnau JL Vanderkooi JM Kent SB 《Journal of the American Chemical Society》2008,130(30):9702-9707
The recently discovered glycine-rich snow flea antifreeze protein (sfAFP) has no sequence homology with any known proteins. No experimental structure has been reported for this interesting protein molecule. Here we report the total chemical synthesis of the mirror image forms of sfAFP (i.e., L-sfAFP, the native protein, and D-sfAFP, the native protein's enantiomer). The predicted 81 amino acid residue polypeptide chain of sfAFP contains Cys residues at positions 1, 13, 28, and 43 and was prepared from four synthetic peptide segments by sequential native chemical ligation. After purification, the full-length synthetic polypeptide was folded at 4 degrees C to form the sfAFP protein containing two disulfides. Chemically synthesized sfAFP had the expected antifreeze activity in an ice recrystallization inhibition assay. Mirror image D-sfAFP protein was prepared by the same synthetic strategy, using peptide segments made from d-amino acids, and had an identical but opposite-sign CD spectrum. As expected, D-sfAFP displays the same antifreeze properties as L-sfAFP, because ice presents an achiral surface for sfAFP binding. Facile synthetic access to sfAFP will enable determination of its molecular structure and systematic elucidation of the molecular basis of the antifreeze properties of this unique protein. 相似文献
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Yi‐Chao Huang Chen‐Chen Chen Shuai Gao Ye‐Hai Wang Prof. Hua Xiao Prof. Feng Wang Prof. Chang‐Lin Tian Prof. Yi‐Ming Li 《Chemistry (Weinheim an der Bergstrasse, Germany)》2016,22(22):7623-7628
Native chemical ligation combined with desulfurization has become a powerful strategy for the chemical synthesis of proteins. Here we describe the use of a new thiol additive, methyl thioglycolate, to accomplish one‐pot native chemical ligation and metal‐free desulfurization for chemical protein synthesis. This one‐pot strategy was used to prepare ubiquitin from two or three peptide segments. Circular dichroism spectroscopy and racemic protein X‐ray crystallography confirmed the correct folding of ubiquitin. Our results demonstrate that proteins synthesized chemically by streamlined 9‐fluorenylmethoxycarbonyl (Fmoc) solid‐phase peptide synthesis coupled with a one‐pot ligation–desulfurization strategy can supply useful molecules with sufficient purity for crystallographic studies. 相似文献
5.
Shan Tang Yan‐Yan Si Zhi‐Peng Wang Kun‐Rong Mei Xin Chen Jing‐Yuan Cheng Dr. Ji‐Shen Zheng Prof. Dr. Lei Liu 《Angewandte Chemie (International ed. in English)》2015,54(19):5713-5717
Successive peptide ligation using a one‐pot method can improve the efficiency of protein chemical synthesis. Although one‐pot three‐segment ligation has enjoyed widespread application, a robust method for one‐pot four‐segment ligation had to date remained undeveloped. Herein we report a new one‐pot multisegment peptide ligation method that can be used to condense up to four segments with operational simplicity and high efficiency. Its practicality is demonstrated by the one‐pot four‐segment synthesis of a plant protein, crambin, and a human chemokine, hCCL21. 相似文献
6.
Abhishek Baral Aromal Asokan Valentin Bauer Bruno Kieffer Vladimir Torbeev 《Tetrahedron》2019,75(6):703-708
Chemical composition of tumor suppressor protein p53 is altered via multiple post-translational modifications which modulate its cellular lifetime and interactions with other biomolecules. Here we report total chemical synthesis of a 61-residue form of transactivation domain (TAD) of p53 based on native chemical ligation of three peptide segments. The experiments to characterize its binding to nuclear co-activator binding domain (NCBD) of CREB-binding protein confirmed native-like induced folding upon binding to NCBD. Thus, the synthetic approach described herein can be useful for the preparation of various post-translationally modified analogues of TAD-p53 for further functional biochemical and biophysical studies. 相似文献
7.
Chemical Synthesis of the 20 kDa Heme Protein Nitrophorin 4 by α‐Ketoacid‐Hydroxylamine (KAHA) Ligation
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Dr. Chunmao He Dr. Sameer S. Kulkarni Dr. Frédéric Thuaud Prof. Dr. Jeffrey W. Bode 《Angewandte Chemie (International ed. in English)》2015,54(44):12996-13001
The chemical synthesis of the 184‐residue ferric heme‐binding protein nitrophorin 4 was accomplished by sequential couplings of five unprotected peptide segments using α‐ketoacid‐hydroxylamine (KAHA) ligation reactions. The fully assembled protein was folded to its native structure and coordinated to the ferric heme b cofactor. The synthetic holoprotein, despite four homoserine residues at the ligation sites, showed identical properties to the wild‐type protein in nitric oxide binding and nitrite dismutase reactivity. This work establishes the KAHA ligation as a valuable and viable approach for the chemical synthesis of proteins up to 20 kDa and demonstrates that it is well‐suited for the preparation of hydrophobic protein targets. 相似文献
8.
Valiyaveetil FI MacKinnon R Muir TW 《Journal of the American Chemical Society》2002,124(31):9113-9120
In this contribution we describe the semisynthesis of the potassium channel, KcsA. A truncated form of KcsA, comprising the first 125 amino acids of the 160-amino acid protein, was synthesized using expressed protein ligation. This truncated form corresponds to the entire membrane-spanning region of the protein and is similar to the construct previously used in crystallographic studies on the KcsA protein. The ligation reaction was carried out using an N-terminal recombinant peptide alpha-thioester, corresponding to residues 1-73 of KcsA, and a synthetic C-terminal peptide corresponding to residues 74-125. Chemical synthesis of the C-peptide was accomplished by optimized Boc-SPPS techniques. A dual fusion strategy, involving glutathione-S-transferase (GST) and the GyrA intein, was developed for recombinant expression of the N-peptide alpha-thioester. The fusion protein, expressed in the insoluble form as inclusion bodies, was refolded and then cleaved successively to remove the GST tag and the intein, thereby releasing the N-peptide alpha-thioester. Following chemical ligation, the KcsA polypeptide was folded into the tetrameric state by incorporation into lipid vesicles. The correctness of the folded state was verified by the ability of the KcsA tetramer to bind to agitoxin-2. To our knowledge, this work represents the first reported semisynthesis of a polytopic membrane protein and highlights the potential application of native chemical ligation and expressed protein ligation for the (semi)synthesis of integral membrane proteins. 相似文献
9.
Javier Sueiras-Diaz Ying Zhang Anastasia Velentza Buyung Santoso Suijin Yang 《Tetrahedron letters》2017,58(25):2448-2455
We report herein, for the first time, a sequential total chemical synthesis of the Human Growth Hormone analog [Nle14,125,170,Glu29,91,Gln74,Asn107,Asp109]hGH-NH2, composed of a 191 amino acid residue polypeptide chain containing two disulfide bonds and nine modifications in the natural sequence. Sequential native chemical ligation of three discrete segments of 52, 52 and 87 amino acid residues gave the target full-length polypeptide chain. Subsequent folding with concomitant formation of the native disulfide bonds afforded a correctly folded homogeneous analog which is biologically active. 相似文献
10.
Rebecca Notis Dardashti Dr. Shailesh Kumar Dr. Shawn M. Sternisha Dr. Post Sai Reddy Prof. Brian G. Miller Prof. Norman Metanis 《Chemistry (Weinheim an der Bergstrasse, Germany)》2020,26(22):4952-4957
Despite their biological importance, post-translationally modified proteins are notoriously difficult to produce in a homogeneous fashion by using conventional expression systems. Chemical protein synthesis or semisynthesis offers a solution to this problem; however, traditional strategies often rely on sulfur-based chemistry that is incompatible with the presence of any cysteine residues in the target protein. To overcome these limitations, we present the design and synthesis of γ-selenolysine, a selenol-containing form of the commonly modified proteinogenic amino acid, lysine. The utility of γ-selenolysine is demonstrated with the traceless ligation of the small ubiquitin-like modifier protein, SUMO-1, to a peptide segment of human glucokinase. The resulting polypeptide is poised for native chemical ligation and chemoselective deselenization in the presence of unprotected cysteine residues. Selenolysine's straightforward synthesis and incorporation into synthetic peptides marks it as a universal handle for conjugating any ubiquitin-like modifying protein to its target. 相似文献
11.
Efficient Palladium‐Assisted One‐Pot Deprotection of (Acetamidomethyl)Cysteine Following Native Chemical Ligation and/or Desulfurization To Expedite Chemical Protein Synthesis
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Dr. Suman Kumar Maity Muhammad Jbara Shay Laps Prof. Ashraf Brik 《Angewandte Chemie (International ed. in English)》2016,55(28):8108-8112
The acetamidomethyl (Acm) moiety is a widely used cysteine protecting group for the chemical synthesis and semisynthesis of peptide and proteins. However, its removal is not straightforward and requires harsh reaction conditions and additional purification steps before and after the removal step, which extends the synthetic process and reduces the overall yield. To overcome these shortcomings, a method for rapid and efficient Acm removal using PdII complexes in aqueous medium is reported. We show, for the first time, the assembly of three peptide fragments in a one‐pot fashion by native chemical ligation where the Acm moiety was used to protect the N‐terminal Cys of the middle fragment. Importantly, an efficient synthesis of the ubiquitin‐like protein UBL‐5, which contains two native Cys residues, was accomplished through the one‐pot operation of three key steps, namely ligation, desulfurization, and Acm deprotection, highlighting the great utility of the new approach in protein synthesis. 相似文献
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本文综述了多肽和蛋白质合成中的片段连接方法,这是近年来多肽和蛋白质合成领域中方法学上的重要进展.该方法使用非保护的多肽片段,无需酶或化学活化试剂,在缓冲溶液中能够高产率地获得多肽和蛋白质.还介绍了与多肽片段连接有关的肽硫酯和肽醛的合成方法. 相似文献
13.
Native Chemical Ligation to Minimize Aspartimide Formation during Chemical Synthesis of Small LDLa Protein
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Dr. Julien Tailhades Ashish Sethi Dr. Emma J. Petrie Prof. Dr. Paul R. Gooley Prof. Dr. Ross A. Bathgate Prof. Dr. John D. Wade Dr. Mohammed A. Hossain 《Chemistry (Weinheim an der Bergstrasse, Germany)》2016,22(3):1146-1151
The inhibition of the G protein‐coupled receptor, relaxin family peptide receptor 1 (RXFP1), by a small LDLa protein may be a potential approach for prostate cancer treatment. However, it is a significant challenge to chemically produce the 41‐residue and three‐disulfide cross‐bridged LDLa module which is highly prone to aspartimide formation due to the presence of several aspartic acid residues. Known palliative measures, including addition of HOBt to piperidine for Nα‐deprotection, failed to completely overcome this side reaction. For this reason, an elegant native chemical ligation approach was employed in which two segments were assembled for generating the linear LDLa protein. Acquisition of correct folding was achieved by using either a regioselective disulfide bond formation or global oxidation strategies. The final synthetic LDLa protein obtained was characterized by NMR spectroscopic structural analysis after chelation with a Ca2+ ion and confirmed to be equivalent to the same protein obtained by recombinant DNA production. 相似文献
14.
Dr. Yasuhiro Kajihara Yasutaka Tanabe Shun Sasaoka Dr. Ryo Okamoto 《Chemistry (Weinheim an der Bergstrasse, Germany)》2012,18(19):5944-5953
The N‐glycosylation of proteins is generated at the consensus sequence NXS/T (where X is any amino acid except proline) by the biosynthetic process, and occurs in the endoplasmic reticulum and Golgi apparatus. In order to investigate the influence of human complex‐type oligosaccharides on counterpart protein conformation, crambin and ovomucoide, which consist of 46 and 56 amino acid residues, respectively, were selected for synthesis of model glycoproteins. These small glycoproteins were intentionally designed to be glycosylated at the α‐helix (crambin: 8 position), β‐sheet (crambin: 2 position) and loop position between the antiparallel β‐sheets (ovomucoide: 28 position), and were synthesized by using a peptide‐segment coupling strategy. After preparation of these glycosylated polypeptide chains, protein folding experiments were performed under redox conditions by using cysteine–cystine. Although the small glycoproteins bearing intentional glycosylation at the α‐helix and β‐sheet exhibited a suitable folding process, glycosylation at the loop position between the antiparallel β‐strands caused multiple products. The conformational differences in the isolated homogeneous glycoproteins compared with non‐glycosylated counterparts were evaluated by circular dichroism (CD) and NMR spectroscopy. These analyses suggested that this intentional N‐glycosylation did not result in large conformational changes in the purified protein structures, including the case of glycosylation at the loop position between the antiparallel β‐strands. In addition to these experiments, the conformational properties of three glycoproteins were evaluated by CD spectroscopy under different temperatures. The oligosaccharides on the protein surface fluctuated considerably; this was dependent on the increase in the solution temperature and was thought to disrupt the protein tertiary structure. Based on the measurement of the CD spectra, however, the glycoproteins bearing three disulfide bonds did not exhibit any change in their protein tertiary structure. These results suggest that the oligosaccharide conformational fluctuations were not disruptive to protein tertiary structure, and the tertiary structure of glycoproteins might be stabilized by the disulfide bond network. 相似文献
15.
Disulfide-reduced form of IL-5 is assembled from three peptide segments in the N to C direction. Reconstitution of the protein under different folding conditions has also been investigated. 相似文献
16.
Baochang Zhang Yupeng Zheng Guochao Chu Xiangyu Deng Tongyue Wang Weiwei Shi Yongkang Zhou Prof. Shan Tang Prof. Ji-Shen Zheng Prof. Lei Liu 《Angewandte Chemie (International ed. in English)》2023,62(33):e202306270
Membrane-associated D-proteins are an important class of synthetic molecules needed for D-peptide drug discovery, but their chemical synthesis using canonical ligation methods such as native chemical ligation is often hampered by the poor solubility of their constituent peptide segments. Here, we describe a B ackbone- I nstalled S plit I ntein- A ssisted L igation (BISIAL) method for the synthesis of these proteins, wherein the native L-forms of the N- and C-intein fragments of the unique consensus-fast (Cfa) (i.e. L–CfaN and L–CfaC) are separately installed onto the two D-peptide segments to be ligated via a removable backbone modification. The ligation proceeds smoothly at micromolar (μM) concentrations under strongly chaotropic conditions (8.0 M urea), and the subsequent removal of the backbone modification groups affords the desired D-proteins without leaving any “ligation scar” on the products. The effectiveness and practicality of the BISIAL method are exemplified by the synthesis of the D-enantiomers of the extracellular domains of T cell immunoglobulin and ITIM domain (TIGIT) and tropomyosin receptor kinase C (TrkC). The BISIAL method further expands the chemical protein synthesis ligation toolkit and provides practical access to challenging D-protein targets. 相似文献
17.
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. 相似文献
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Manfred Mutter Karl-Heinz Altmann Klaus Müller StPhane Vuilleumier Thomas Vorherr 《Helvetica chimica acta》1986,69(5):985-995
A general procedure for the design of synthetic vaccines with the retained conformational features of protein antigenic determinants is described. This new concept emerges from detailed studies on the relationship between primary sequence and secondary structure formation of synthetic peptides and takes advantage of the amphiphilic nature of epitope-containing peptide segments in the native protein to accomplish structural modifications. These segments, for example amphiphilic helices or β-sheets, are stabilized by the insertion of secondary structure-inducing amino-acid residues on the hydrophobic part of the peptide without affecting the spatial arrangement of functional residues on the hydrophilic side. The availability of amphiphilic peptides with tailor-made conformational properties, e.g. helices, β-sheets, and, moreover, assemblies of these blocks to structures of higher order (‘folding units’), allows the presentation and stabilization of continuous as well as discontinuous epitopes by this approach. This strategy is exemplified for the case of two discontinuous epitopes taken from lysozyme, which are matched to host molecules with adequate conformational features by the help of computer-assisted molecular modelling. The implications of this new concept for the design of synthetic vaccines are discussed with special emphasis to the important role of peptide synthesis and chemical structure modification. 相似文献
20.
Cyclotides constitute a fascinating family of circular proteins containing ca.30 amino acid residues.They have a unique cyclic cysteine knot topology and exhibit remarkable thermal,chemical and enzymatic stabilities.These characteristics enable them to have a range of biological activities and promising pharmaceutical and agricultural applications.Here,we present a practical strategy for the chemical synthesis of cyclotides through the intramolecular ligation of fully unprotected peptide O-esters.This strategy involves the mild Fmoc solid-phase peptide synthesis of the peptide O-ester backbone,the head-to-tail cyclization of the cyclotide backbone by native chemical ligation,and the oxidative refolding to yield the natural knot protein.The simplicity and high efficiency of the strategy can be employed in the synthesis of artificial cyclotides for pharmaceutical applications. 相似文献