共查询到20条相似文献,搜索用时 15 毫秒
1.
Atoosa Rezvanpour Jeremy M. Phillips Gary S. Shaw 《Protein science : a publication of the Protein Society》2009,18(12):2528-2536
S100B and S100A10 are dimeric, EF‐hand proteins. S100B undergoes a calcium‐dependant conformational change allowing it to interact with a short contiguous sequence from the actin‐capping protein CapZ (TRTK12). S100A10 does not bind calcium but is able to recruit the N‐terminus of annexin A2 important for membrane fusion events, and to form larger multiprotein complexes such as that with the cation channel proteins TRPV5/6. In this work, we have designed, expressed, purified, and characterized two S100‐target peptide hybrid proteins comprised of S100A10 and S100B linked in tandem to annexin A2 (residues 1–15) and CapZ (TRTK12), respectively. Different protease cleavage sites (tobacco etch virus, PreScission) were incorporated into the linkers of the hybrid proteins. In situ proteolytic cleavage monitored by 1H‐15N HSQC spectra showed the linker did not perturb the structures of the S100A10‐annexin A2 or S100B‐TRTK12 complexes. Furthermore, the analysis of the chemical shift assignments (1H, 15N, and 13C) showed that residues T102‐S108 of annexin A2 formed a well‐defined α‐helix in the S100A10 hybrid while the TRTK12 region was unstructured at the N‐terminus with a single turn of α‐helix from D108‐K111 in the S100B hybrid protein. The two S100 hybrid proteins provide a simple yet extremely efficient method for obtaining high yields of intact S100 target peptides. Since cleavage of the S100 hybrid protein is not necessary for structural characterization, this approach may be useful as a scaffold for larger S100 complexes. 相似文献
2.
David S. Waugh 《Protein science : a publication of the Protein Society》2016,25(3):559-571
Although chaperone‐assisted protein crystallization remains a comparatively rare undertaking, the number of crystal structures of polypeptides fused to maltose‐binding protein (MBP) that have been deposited in the Protein Data Bank (PDB) has grown dramatically during the past decade. Altogether, 102 fusion protein structures were detected by Basic Local Alignment Search Tool (BLAST) analysis. Collectively, these structures comprise a range of sizes, space groups, and resolutions that are typical of the PDB as a whole. While most of these MBP fusion proteins were equipped with short inter‐domain linkers to increase their rigidity, fusion proteins with long linkers have also been crystallized. In some cases, surface entropy reduction mutations in MBP appear to have facilitated the formation of crystals. A comparison of the structures of fused and unfused proteins, where both are available, reveals that MBP‐mediated structural distortions are very rare. 相似文献
3.
Robert E. Thompson Tom W. Muir 《Protein science : a publication of the Protein Society》2018,27(3):614-619
Naturally split inteins mediate a traceless protein ligation process known as protein trans‐splicing (PTS). Although frequently used in protein engineering applications, the efficiency of PTS can be reduced by the tendency of some split intein fusion constructs to aggregate; a consequence of the fragmented nature of the split intein itself or the polypeptide to which it is fused (the extein). Here, we report a strategy to help address this liability. This involves embedding the split intein within a protein sequence designed to stabilize either the intein fragment itself or the appended extein. We expect this approach to increase the scope of PTS‐based protein engineering efforts. 相似文献
4.
Byungsu Kwon Daniel Tietze Paul B. White Shu Y. Liao Mei Hong 《Protein science : a publication of the Protein Society》2015,24(7):1087-1099
Solid‐state NMR‐based structure determination of membrane proteins and large protein complexes faces the challenge of limited spectral resolution when the proteins are uniformly 13C‐labeled. A strategy to meet this challenge is chemical ligation combined with site‐specific or segmental labeling. While chemical ligation has been adopted in NMR studies of water‐soluble proteins, it has not been demonstrated for membrane proteins. Here we show chemical ligation of the influenza M2 protein, which contains a transmembrane (TM) domain and two extra‐membrane domains. The cytoplasmic domain, which contains an amphipathic helix (AH) and a cytoplasmic tail, is important for regulating virus assembly, virus budding, and the proton channel activity. A recent study of uniformly 13C‐labeled full‐length M2 by spectral simulation suggested that the cytoplasmic tail is unstructured. To further test this hypothesis, we conducted native chemical ligation of the TM segment and part of the cytoplasmic domain. Solid‐phase peptide synthesis of the two segments allowed several residues to be labeled in each segment. The post‐AH cytoplasmic residues exhibit random‐coil chemical shifts, low bond order parameters, and a surface‐bound location, thus indicating that this domain is a dynamic random coil on the membrane surface. Interestingly, the protein spectra are similar between a model membrane and a virus‐mimetic membrane, indicating that the structure and dynamics of the post‐AH segment is insensitive to the lipid composition. This chemical ligation approach is generally applicable to medium‐sized membrane proteins to provide site‐specific structural constraints, which complement the information obtained from uniformly 13C, 15N‐labeled proteins. 相似文献
5.
Stephen Kent Youhei Sohma Suhuai Liu Duhee Bang Brad Pentelute Kalyaneswar Mandal 《Journal of peptide science》2012,18(7):428-436
‘Chemical ligation’ – the regioselective and chemoselective covalent condensation of unprotected peptide segments – has enabled the synthesis of polypeptide chains of more than 200 amino acids. An efficient total chemical synthesis of the insulin molecule has been devised on the basis of a key ester‐linked intermediate that is chemically converted to fully active human insulin. Enzyme molecules of defined covalent structure and with full enzymatic activity have been prepared and characterized by high‐resolution X‐ray crystallography. A ‘glycoprotein mimetic’ of defined chemical structure and with a mass of 50,825 Da, has been prepared and shown to have full biological activity and improved pharmacokinetic properties. d ‐Protein molecules that are the mirror images of proteins found in the natural world have been prepared by total chemical synthesis. Racemic protein mixtures, consisting of the d ‐enantiomers and l ‐enantiomers of a protein molecule, form highly ordered centrosymmetric crystals with great ease; this has enabled the determination of the crystal structures of recalcitrant protein molecules. A protein with a novel linear‐loop covalent topology of the peptide chain has been designed and synthesized and its structure determined by facile crystallization as the quasi‐racemate with the d ‐form of the native protein molecule. We have developed an optimized total chemical synthesis of biologically active vascular endothelial growth factor‐A; total synthesis of the mirror‐image protein will be used to systematically develop d ‐protein antagonists of this important growth factor. The total chemical synthesis of proteins is now a practical reality and enables access to a new world of protein molecules. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
6.
Franziska Thomas 《Journal of peptide science》2013,19(3):141-147
The chemical synthesis of proteins has facilitated functional studies of proteins due to the site‐specific incorporation of post‐translational modifications, labels, and non‐proteinogenic amino acids. Moreover, native chemical ligation provides facile access to proteins by chemical means. However, the application of the native chemical ligation reaction in the synthesis of parallel formats such as protein arrays has been complicated because of the often cumbersome and time‐consuming synthesis of the required peptide thioesters. An Fmoc‐based peptide thioester synthesis with self‐purification on the sulfonamide ‘safety‐catch’ linker widens this bottleneck because HPLC purification can be avoided. The method is based on an on‐resin cyclization–thiolysis reaction sequence. A macrocyclization via the N‐terminus of the full‐length peptide followed by a thiolytic C‐terminal ring opening allows selective detachment of the truncation products and the full‐length peptide. A brief overview of the chemical aspects of this method is provided including the optimization steps and the automation process. Furthermore, the application of the cyclization–thiolysis approach combined with the native chemical ligation reaction in the parallel synthesis of a library of 16 SH3‐domain variants of SHO1 in yeast is described, demonstrating the value of this new technique for the chemical synthesis of protein arrays. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. 相似文献
7.
Tagged library approach to chemical genomics and proteomics 总被引:1,自引:0,他引:1
Proteomics and chemical genomics face great challenges in the form of molecular libraries of ever increasing size and diversity requiring rapid screening, coupled with a growing number of target proteins for which complimentary molecular ligands are sought. Proteomics and chemical genomics are at a stage that requires techniques which can dramatically accelerate the discovery process. One technique that has shown great promise in accomplishing this is the tagged library approach. It entails the synthetic inclusion of an internal tag from the beginning of the synthesis. This tag adds another degree of functionality to the molecule, in addition to mere ligation, that eliminates the need for time-consuming steps downstream in the process. The tag's functional possibilities span a variety of uses including internal fluorophores, intrinsic binding motifs that enable compound identification, functionalities that play the major role in the synthesis of the ligand itself, and internal linkers that eliminate the need for lengthy 'tether effect' structure-activity relationship studies. 相似文献
8.
In planta chemical cross‐linking and mass spectrometry analysis of protein structure and interaction in Arabidopsis
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Site‐specific chemical cross‐linking in combination with mass spectrometry analysis has emerged as a powerful proteomic approach for studying the three‐dimensional structure of protein complexes and in mapping protein–protein interactions (PPIs). Building on the success of MS analysis of in vitro cross‐linked proteins, which has been widely used to investigate specific interactions of bait proteins and their targets in various organisms, we report a workflow for in vivo chemical cross‐linking and MS analysis in a multicellular eukaryote. This approach optimizes the in vivo protein cross‐linking conditions in Arabidopsis thaliana, establishes a MudPIT procedure for the enrichment of cross‐linked peptides, and develops an integrated software program, exhaustive cross‐linked peptides identification tool (ECL), to identify the MS spectra of in planta chemical cross‐linked peptides. In total, two pairs of in vivo cross‐linked peptides of high confidence have been identified from two independent biological replicates. This work demarks the beginning of an alternative proteomic approach in the study of in vivo protein tertiary structure and PPIs in multicellular eukaryotes. 相似文献
9.
Frank Sainsbury Philippe Varennes‐Jutras Marie‐Claire Goulet Marc‐André D'Aoust Dominique Michaud 《Plant biotechnology journal》2013,11(9):1058-1068
Studies have reported the usefulness of fusion proteins to bolster recombinant protein yields in plants. Here, we assess the potential of tomato SlCYS8, a Cys protease inhibitor of the cystatin protein superfamily, as a stabilizing fusion partner for human alpha‐1‐antichymotrypsin (α1ACT) targeted to the plant cell secretory pathway. Using the model expression platform Nicotiana benthamiana, we show that the cystatin imparts a strong stabilizing effect when expressed as a translational fusion with α1ACT, allowing impressive accumulation yields of over 2 mg/g of fresh weight tissue for the human serpin, a 25‐fold improvement on the yield of α1ACT expressed alone. Natural and synthetic peptide linkers inserted between SlCYS8 and α1ACT have differential effects on protease inhibitory potency of the two protein partners in vitro. They also have a differential impact on the yield of α1ACT, dependent on the extent to which the hybrid protein may remain intact in the plant cell environment. The stabilizing effect of SlCYS8 does not involve Cys protease inhibition and can be partly reproduced in the cytosol, where peptide linkers are less susceptible to degradation. The effect of SlCYS8 on α1ACT yields could be explained by: (i) an improved translation of the human protein coding sequence; and/or (ii) an overall stabilization of its tertiary structure preventing proteolytic degradation and/or polymerization. These findings suggest the potential of plant cystatins as stabilizing fusion partners for recombinant proteins in plant systems. They also underline the need for an empirical assessment of peptide linker functions in plant cell environments. 相似文献
10.
Plonsky I Kingsley DH Rashtian A Blank PS Zimmerberg J 《Biology of the cell / under the auspices of the European Cell Biology Organization》2008,100(6):377-386
Background information. Protein‐mediated merger of biological membranes, membrane fusion, is an important process. To investigate the role of fusogenic proteins in the initial size and dynamics of the fusion pore (a narrow aqueous pathway, which widens to finalize membrane fusion), two different fusion proteins expressed in the same cell line were investigated: the major glycoprotein of baculovirus Autographa californica (GP64) and the HA (haemagglutinin) of influenza X31. Results. The host Sf9 cells expressing these viral proteins, irrespective of protein species, fused to human RBCs (red blood cells) upon acidification of the medium. A high‐time‐resolution electrophysiological study of fusion pore conductance revealed fundamental differences in (i) the initial pore conductance; pores created by HA were smaller than those created by GP64; (ii) the ability of pores to flicker; only HA‐mediated pores flickered; and (iii) the time required for pore formation; HA‐mediated pores took much longer to form after acidification. Conclusion. HA and GP64 have divergent electrophysiological phenotypes even when they fuse identical membranes, and fusion proteins play a crucial role in determining initial fusion pore characteristics. The structure of the initial fusion pore detected by electrical conductance measurements is sensitive to the nature of the fusion protein. 相似文献
11.
Vishnu Priyanka Reddy Chichili Veerendra Kumar J. Sivaraman 《Protein science : a publication of the Protein Society》2013,22(2):153-167
Linkers or spacers are short amino acid sequences created in nature to separate multiple domains in a single protein. Most of them are rigid and function to prohibit unwanted interactions between the discrete domains. However, Gly‐rich linkers are flexible, connecting various domains in a single protein without interfering with the function of each domain. The advent of recombinant DNA technology made it possible to fuse two interacting partners with the introduction of artificial linkers. Often, independent proteins may not exist as stable or structured proteins until they interact with their binding partner, following which they gain stability and the essential structural elements. Gly‐rich linkers have been proven useful for these types of unstable interactions, particularly where the interaction is weak and transient, by creating a covalent link between the proteins to form a stable protein–protein complex. Gly‐rich linkers are also employed to form stable covalently linked dimers, and to connect two independent domains that create a ligand‐binding site or recognition sequence. The lengths of linkers vary from 2 to 31 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked partners. Various structures of covalently linked protein complexes have been described using X‐ray crystallography, nuclear magnetic resonance and cryo‐electron microscopy techniques. In this review, we evaluate several structural studies where linkers have been used to improve protein quality, to produce stable protein–protein complexes, and to obtain protein dimers. 相似文献
12.
13.
Co-stimulatory molecules can be efficiently produced as a recombinant protein in E. coli with a large range of applications in the fields of immunotherapy. However, whether, different fusions that would affect
their functions have rarely been analyzed. To explore the effects of different fusions and linkers on the molecular conformation
and activity of CD137 ligand (CD137L), a recombinant human CD137L protein (rhCD137L) library, which consists of the entire
extracellular domain of human CD137L fused to N- or C-terminal His-tag through different linkers, was constructed and all
rhCD137Ls were, respectively, expressed in E. coli BL21 (DE3) strain carrying a chaperone plasmid pG-Tf2. After purification of the soluble rhCD137Ls, the recombinant fusion
proteins could markedly promote the growth of activated T cells, but their effects on cytokine productions were different
from each other. The present work indicated that, although all rhCD137Ls have desired biological activity, different fusions
and linkers did affect their structures and functions. Consequently, rational design of a library is a necessary and feasible
approach for fusion proteins in order to obtain a satisfactory drug candidate for further development. 相似文献
14.
A truncated and dimeric format of an Affibody library on bacteria enables FACS‐mediated isolation of amyloid‐beta aggregation inhibitors with subnanomolar affinity
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The amyloid hypothesis suggests that accumulation of amyloid β (Aβ) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aβ aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Aβ peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Aβ peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high‐affinity Aβ‐binding molecules were isolated using flow‐cytometric sorting. The best performing candidate binds Aβ with a KD value of around 300 pM, corresponding to a 50‐fold improvement in affinity relative to the first‐generation binder. The new dimeric Affibody molecule was shown to capture Aβ1‐42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide. 相似文献
15.
Na Sun Noriko Tamura Tomohiro Tamura Roland Wilhelm Knispel Thomas Hrabe Christine Kofler Stephan Nickell István Nagy 《Proteomics》2009,9(14):3783-3786
We used molecular sieve chromatography in combination with LC‐MS/MS to identify protein complexes that can serve as templates in the template matching procedures of visual proteomics approaches. By this method the sample complexity was lowered sufficiently to identify 464 proteins and – on the basis of size distribution and bioinformatics analysis – 189 of them could be assigned as subunits of macromolecular complexes over the size of 300 kDa. From these we purified six stable complexes of Thermoplasma acidophilum whose size and subunit composition – analyzed by electron microscopy and MALDI‐TOF‐MS, respectively – verified the accuracy of our method. 相似文献
16.
Cristina Banfi Dr. Maura Brioschi Simona Barcella Robin Wait Shajna Begum Sabrina Galli Andrea Rizzi Elena Tremoli 《Proteomics》2009,9(5):1344-1352
The molecular mechanisms underlying the relationship between low‐density lipoprotein (LDL) and the risk of atherosclerosis are not clear. Therefore, detailed information on the protein composition of LDL may help to reveal its role in atherogenesis. Liquid‐phase IEF has been used to resolve LDL proteins into well‐defined fractions on the basis of pI, which improves the subsequent detection and resolution of low abundance proteins. Besides known LDL‐associated proteins, this approach revealed the presence of proteins not previously described to reside in LDL, including prenylcysteine lyase (PCL1), orosomucoid, retinol‐binding protein, and paraoxonase‐1. PCL1, an enzyme crucial for the degradation of prenylated proteins, generates free cysteine, isoprenoid aldehyde and hydrogen peroxide. Addition of the substrate farnesylcysteine to lipoprotein resulted in a time‐dependent generation of H2O2 which was stronger in very low density lipoprotein (VLDL) than in LDL or HDL, reflecting the greater protein content of PCL1 in VLDL. Farnesol, a dead end inhibitor of the PCL1 reaction, reduced H2O2 generation by VLDL. PCL1 is generated along with nascent lipoprotein, as shown by its presence in the lipoprotein secreted by HepG2 cells. The finding that an enzyme associated with atherogenic lipoproteins can itself generate an oxidant suggests that PCL1 may play a significant role in atherogenesis. 相似文献
17.
miR‐143 targets MAPK7 in CHO cells and induces a hyperproductive phenotype to enhance production of difficult‐to‐express proteins
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Melanie Schoellhorn Simon Fischer Andreas Wagner René Handrick Kerstin Otte 《Biotechnology progress》2017,33(4):1046-1058
In recent years, the number of complex but clinically effective biologicals such as multi‐specific antibody formats and fusion proteins has increased dramatically. However, compared to classical monoclonal antibodies (mAbs), these rather artificially designed therapeutic proteins have never undergone millions of years of evolution and thus often turn out to be difficult‐to‐express using mammalian expression systems such as Chinese hamster ovary (CHO) cells. To provide access to these sophisticated but effective drugs, host cell engineering of CHO production cell lines represents a promising approach to overcome low production yields. MicroRNAs (miRNAs) have recently gained much attention as next‐generation cell engineering tools. However, only very little is known about the capability of miRNAs to specifically increase production of difficult‐to‐express proteins. In a previous study we identified miR‐143 amongst others to improve protein production in CHO cells. Thus, the aim of the present study was to examine if miR‐143 might be suitable to improve production of low yield protein candidates. Both transient and stable overexpression of miR‐143 significantly improved protein production without negatively affecting cell growth and viability of different recombinant CHO cells. In addition, mitogen‐activated protein kinase 7 (MAPK7) was identified as a putative target gene of miR‐143‐3p in CHO cells. Finally, siRNA‐mediated knock‐down of MAPK7 could be demonstrated to phenocopy pro‐productive effects of miR‐143. In summary, our data suggest that miR‐143 might represent a novel genetic element to enhance production of difficult‐to‐express proteins in CHO cells which may be partly mediated by down‐regulation of MAPK7. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1046–1058, 2017 相似文献
18.
Hao Xu Youngsoo Jun James Thompson John Yates William Wickner 《The EMBO journal》2010,29(12):1948-1960
SNARE‐dependent membrane fusion requires the disassembly of cis‐SNARE complexes (formed by SNAREs anchored to one membrane) followed by the assembly of trans‐SNARE complexes (SNAREs anchored to two apposed membranes). Although SNARE complex disassembly and assembly might be thought to be opposing reactions, the proteins promoting disassembly (Sec17p/Sec18p) and assembly (the HOPS complex) work synergistically to support fusion. We now report that trans‐SNARE complexes formed during vacuole fusion are largely associated with Sec17p. Using a reconstituted proteoliposome fusion system, we show that trans‐SNARE complex, like cis‐SNARE complex, is sensitive to Sec17p/Sec18p mediated disassembly. Strikingly, HOPS inhibits the disassembly of SNARE complexes in the trans‐, but not in the cis‐, configuration. This selective HOPS preservation of trans‐SNARE complexes requires HOPS:SNARE recognition and is lost when the apposed bilayers are dissolved in Triton X‐100; it is also observed during fusion of isolated vacuoles. HOPS thus directs the Sec17p/Sec18p chaperone system to maximize functional trans‐SNARE complex for membrane fusion, a new role of tethering factors during membrane traffic. 相似文献
19.
Baptiste Leroy Caroline Rosier Vanessa Erculisse Natalie Leys Max Mergeay Ruddy Wattiez 《Proteomics》2010,10(12):2281-2291
Among differential proteomic methods based on stable isotopic labeling, isotope‐coded protein labeling (ICPL) is a recent non‐isobaric technique devised to label primary amines found in proteins. ICPL overcomes some of the disadvantages found in other chemical‐labeling techniques, such as iTRAQ or ICAT. However, previous analyses revealed that more than 30% of the proteins identified in regular ICPL generally remain unquantified. In this study, we describe a modified version of ICPL, named Post‐digest ICPL, that makes it possible to label and thus to quantify all the peptides in a sample (bottom–up approach). Optimization and validation of this Post‐digest ICPL approach were performed using a standard protein mixture and complex protein samples. Using this strategy, the number of proteins that were identified and quantified was greatly increased in comparison with regular ICPL and cICAT approaches. The pros and cons of this improvement are discussed. This complementary approach to traditional ICPL was applied to the analysis of modification of protein abundances in the model bacterium Cupriavidus metallidurans CH34 after cultivation under simulated microgravity. In this context, two different systems – a 2‐D clinorotation and 3‐D random positioning device – were used and the results were compared and discussed. 相似文献
20.
Isabel Pardo Ana Isabel Vicente Diana M. Mate Miguel Alcalde Susana Camarero 《Biotechnology and bioengineering》2012,109(12):2978-2986
DNA recombination methods are useful tools to generate diversity in directed evolution protein engineering studies. We have designed an array of chimeric laccases with high‐redox potential by in vitro and in vivo DNA recombination of two fungal laccases (from Pycnoporus cinnabarinus and PM1 basidiomycete), which were previously tailored by laboratory evolution for functional expression in Saccharomyces cerevisiae. The laccase fusion genes (including the evolved α‐factor prepro‐leaders for secretion in yeast) were subjected to a round of family shuffling to construct chimeric libraries and the best laccase hybrids were identified in dual high‐throughput screening (HTS) assays. Using this approach, we identified chimeras with up to six crossover events in the whole sequence, and we obtained active hybrid laccases with combined characteristics in terms of pH activity and thermostability. Biotechnol. Bioeng. 2012; 109: 2978–2986. © 2012 Wiley Periodicals, Inc. 相似文献