Tuesday, January 17, 2012

peptide solubility | What is peptide solubility|Papers on peptide solubility|Research on peptide solubility | Publications on peptide solubility


1.
J Sci Food Agric. 2012 Jan 13. doi: 10.1002/jsfa.5573. [Epub ahead of print]

Hypocholesterolaemic and antioxidant activities of chickpea (Cicer arietinum L.) protein hydrolysates.

Source

Instituto de la Grasa-CSIC, Av. Padre García Tejero, 4, 41012-Seville, Spain. mdmar@cica.es.

Abstract

BACKGROUND:

Some dietary proteins possess biological properties which make them potential ingredients of functional or health-promoting foods. Many of these properties are attributed to bioactive peptides that can be released by controlled hydrolysis using exogenous proteases. The aim of this work was to test the improvement of hypocholesterolaemic and antioxidant activities of chickpea protein isolate by means of hydrolysis with alcalase and flavourzyme.

RESULTS:

All hydrolysates tested exhibited better hypocholesterolaemic activity when compared with chickpea protein isolate. The highest cholesterol micellar solubility inhibition (50%) was found after 60 min of treatment with alcalase followed by 30 min of hydrolysis with flavourzyme. To test antioxidant activity of chickpea proteins three methods were used: β-carotene bleaching method, reducing power and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect since antioxidant activity of protein hydrolysates may not be attributed to a single mechanism. Chickpea hydrolysates showed better antioxidant activity in all assays, especially reducing power and DPPH scavenging effect than chickpea protein isolate.

CONCLUSION:

The results of this study showed the good potential of chickpea protein hydrolysates as bioactive ingredients. The highest bioactive properties could be obtained by selecting the type of proteases and the hydrolysis time. Copyright © 2012 Society of Chemical Industry.

Copyright © 2012 Society of Chemical Industry.

PMID:
22246802
[PubMed - as supplied by publisher]
2.
Bioconjug Chem. 2012 Jan 13. [Epub ahead of print]

Nanomolar cellular antisense activity of peptide nucleic acid (PNA) cholic acid ("umbrella") and cholesterol conjugates delivered by cationic lipids.

Abstract

Limited cellular uptake and low bioavailability of peptide nucleic acids (PNAs) have restricted widespread use of PNAs as antisense/antigene agents for cells in culture and not least for in vivo applications. We now report the synthesis and cellular antisense activity in cultured HeLa pLuc705 cells of cholesterol and cholic acid ("umbrella") derivatives of splice correction antisense PNA oligomers. While the conjugates alone were practically inactive up to 1 μM, their activity was dramatically improved when delivered by a cationic lipid transfection agent (LipofectAMINE2000). In particular, PNAs, conjugated to cholesterol through an ester hemisuccinate linker or to cholic acid exhibited low nanomolar activity (EC50 ~ 25 nM). Excellent sequence specificity was retained as mismatch PNA conjugates did not show any significant antisense activity. Furthermore, we show that increasing the transfection volume improved transfection efficiency suggesting that accumulation (condensation) of the PNA/lipid complex on the cellular surface is part of the uptake mechanism. These results provide a novel, simple method for very efficient cellular delivery of PNA oligomers, especially using PNA-cholic acid conjugates which, in contrast to PNA-cholesterol conjugates exhibit sufficient water solubility. The results also question the generality of using cholic acid "umbrella" derivatives as a delivery modality for antisense oligomers.

PMID:
22243634
[PubMed - as supplied by publisher]
3.
J Mol Biol. 2012 Jan 2. [Epub ahead of print]

The Antibiotic Thermorubin Inhibits Protein Synthesis by Binding to Inter-Subunit Bridge B2a of the Ribosome.

Source

Department of Chemistry, Yale University, 225 Prospect Street, PO Box 208107, New Haven, CT 06520-8107, USA.

Abstract

Thermorubin is a small-molecule inhibitor of bacterial protein synthesis, but relatively little is known about the molecular mechanism by which it blocks translation. The structure of the complex between thermorubin and the 70S ribosome from Thermus thermophilus reported here shows that thermorubin interacts with the ribosome in a way that is distinct from any other known class of ribosome inhibitor. Though it is structurally similar to tetracycline, it binds to the ribosome at an entirely different location-the interface between the small and large subunits that is formed by inter-subunit bridge B2a. This region of the ribosome is known to play a role in the initiation of translation, and thus, the binding site we observe is consistent with evidence suggesting that thermorubin inhibits the initiation stage of protein synthesis. The binding of thermorubin induces a rearrangement of two bases on helix 69 of the 23S rRNA, and presumably, this rearrangement blocks the binding of an A-site tRNA, thereby inhibiting peptide bond formation. Due in part to its low solubility in aqueous media, thermorubin has not been used clinically, although it is a potent antibacterial agent with low toxicity (Therapeutic Index>200). The interactions between thermorubin and the ribosome, as well as its adjacency to the observed binding sites of three other antibiotic classes, may enable the design of novel derivatives that share thermorubin's mode of action but possess improved pharmacodynamic properties.

Copyright © 2011. Published by Elsevier Ltd.

PMID:
22240456
[PubMed - as supplied by publisher]
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4.
Chem Biol Drug Des. 2012 Jan 10. doi: 10.1111/j.1747-0285.2012.01324.x. [Epub ahead of print]

Molecular Dynamics in Drug Design: New Generations of Compstatin Analogs.

Source

Department of Bioengineering, University of California, Riverside, California 92521, USA. Department of Physics, University of Cyprus, PO20537, CY1678, Nicosia, Cyprus. Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey 08544, USA.

Abstract

We report the computational and rational design of new generations of several tryptophan-rich peptides from the compstatin family. The binding efficacy of the peptides has been tested using extensive molecular dynamics-based structural and physicochemical analysis, using 32 atomic-detail trajectories in explicit water for 22 peptides bound to human, rat, or mouse target protein C3, to a total of 257 nanoseconds. The criteria for the new designs are: (i) optimization for high binding affinity and for the balance between hydrophobicity and polarity to improve solubilitycompared to known compstatin analogs; and (ii) development of dual specificity anti-human-rat/mouse C3 analogs, which is important for use in animal models for disease, given the species specificity of known compstatin analogs. Three of the new analogs have been analyzed in more detail as they possess strong and novel binding characteristics and are promising candidates for further optimization. This work paves the way for the development of an improved therapeutic for age-related macular degeneration, and other complement system-mediated diseases, compared to known compstatin variants. © 2012 John Wiley & Sons A/S.

© 2012 John Wiley & Sons A/S.

PMID:
22233517
[PubMed - as supplied by publisher]
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5.
Biomaterials. 2012 Jan 6. [Epub ahead of print]

Delivery of membrane impermeable cargo into CHO cells by peptidenanoparticles targeted by a protein corona.

Source

Dept. of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.

Abstract

Nanocarriers can fulfill essential functions in the stabilization and delivery of drugs: they prevent solubility issues and degradation, reduce side effects and modify the pharmacokinetic profile. However, particle based pharmaceuticals are complex and thus challenging to scale up. As formulation routines account for a large fraction of production costs, reducing complexity in the process of assembly, loading and functionalization of nanoparticles is desirable. Unlike existing approaches with similar goals, our protocol is designed to minimize usage of material and time. Prerequisite to this elegant one-step-procedure is the controlled phase-separation of a hydrophobic peptide to nanoparticles, inducing concurrent cargo-entrapment and association of a protein corona. We demonstrate the process by assembling Flutax-2 containing peptide nanoparticles functionalized with transferrin. Cellular uptake of the particles and cargo release depend on specific particle-cell interactions via transferrin receptor. These data indicate corona-mediated delivery of membrane impermeable cargo in vitro by a particulate delivery system entirely composed of amino acids.

Copyright © 2011 Elsevier Ltd. All rights reserved.

PMID:
22226586
[PubMed - as supplied by publisher]
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6.
Microb Cell Fact. 2011 Dec 23;10(1):112. [Epub ahead of print]

The order of expression is a key factor in the production of active transglutaminase in Escherichia coli by co-expression with its pro-peptide.

Abstract

ABSTRACT:

BACKGROUND:

Streptomyces transglutaminase (TGase) is naturally synthesized as zymogen (pro-TGase), which is then processed to produce active enzyme by the removal of its N-terminal pro-peptide. This pro-peptide is found to be essential for overexpression of soluble TGase in E. coli. However, expression of pro-TGase by E. coli requires protease-mediated activation in vitro. In this study, we developed a novel co- expression method for the direct production of active TGase in E. coli.

RESULTS:

A TGase from S. hygroscopicus was expressed in E. coli only after fusing with the pelB signal peptide, but fusion with the signal peptide induced insoluble enzyme. Therefore, alternative protocol was designed by co-expressing the TGase and its pro-peptide as independent polypeptides under a single T7 promoter using vector pET-22b(+). Although the pro-peptide was co-expressed, the TGase fused without the signal peptide was undetectable in both soluble and insoluble fractions of the recombinant cells. Similarly, when both genes were expressed in the order of the TGase and the pro-peptide, the solubility of TGase fused with the signal peptide was not improved by the co-expression with its pro-peptide. Interestingly, active TGase was only produced by the cells in which the pro-peptide and the TGase were fused with the signal peptide and sequentially expressed. The purified recombinant and native TGase shared the similar catalytic properties.

CONCLUSIONS:

Our results indicated that the pro-peptide can assist correct folding of the TGase inter-molecularly in E. coli, and expression of pro-peptide prior to that of TGase was essential for the production of active TGase. The co-expression strategy based on optimizing the order of gene expression could be useful for the expression of other functional proteins that are synthesized as a precursor.

PMID:
22196373
[PubMed - as supplied by publisher]
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7.
Biopolymers. 2011;96(5):688-93.

NMEGylation: a novel modification to enhance the bioavailability of therapeuticpeptides.

Source

Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.

Abstract

We have evaluated "NMEGylation"--the covalent attachment of an oligo-N-methoxyethylglycine (NMEG) chain--as a new form of peptide/protein modification to enhance the bioavailability of short peptides. OligoNMEGs are hydrophilic polyethylene glycol-like molecules made by solid-phase synthesis, typically up to 40 monomers in length. They have been studied as nonfouling surface coatings and as monodisperse mobility modifiers for free-solution conjugate capillary electrophoresis. However, polyNMEGs have not been demonstrated before this work as modifiers of therapeutic proteins. In prior published work, we identified a short peptide, "C20," as a potential extracellular inhibitor of the fusion of human respiratory syncytial virus with mammalian cells. The present study was aimed at improving the C20 peptide's stability and solubility. To this end, we synthesized and studied a series of NMEGylated C20 peptide-peptoid bioconjugates comprising different numbers of NMEGs at either the N- or C-terminus of C20. NMEGylation was found to greatly improve this peptide's solubility and serum stability; however, longer polyNMEGs (n > 3) deleteriously affectedpeptide binding to the target protein. By incorporating just one NMEG monomer, along with a glycine monomer as a flexible spacer, at C20's N-terminus (NMEG-Gly-C20), we increased both solubility and serum stability greatly, while recovering a binding affinity comparable to that of unmodified C20 peptide. Our results suggest that NMEGylation with an optimized number of NMEG monomers and a proper linker could be useful, more broadly, as a novel modification to enhance bioavailability and efficacy of therapeutic peptides.

PMID:
22180913
[PubMed - in process]
PMCID: PMC3243944
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8.
Chembiochem. 2012 Jan 23;13(2):271-81. doi: 10.1002/cbic.201100607. Epub 2011 Dec 15.

Phosphorylation as a tool to modulate aggregation propensity and to predict fibril architecture.

Source

School of Chemistry, University of Leeds, Leeds, LS21 3DS (UK); Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS21 3DS (UK).

Abstract

Despite the importance of post-translational modifications in controlling the solubility and conformational properties of proteins and peptides, precisely how the aggregation propensity of different peptide sequences is modulated by chemical modification remains unclear. Here we have investigated the effect of phosphorylation on the aggregation propensity of a 13-residue synthetic peptide incorporating one or more phosphate groups at seven different sites at various pH values. Fibril formation was shown to be inhibited when a single phosphate group was introduced at all seven locations in the peptide sequence at pH 7.5, when the phosphate group is fully charged. By contrast, when the samepeptides were analysed at pH 1.1, when the phosphate is fully protonated, fibrils from all seven peptide sequences form rapidly. At intermediate pH values (pH 3.6) when the phosphate group is mono-anionic, the aggregation propensity of thepeptides was found to be highly dependent on the position of the phosphate group in the peptide sequence. Using this information, combined with molecular dynamics (MD) simulations of the peptide sequence, we provide evidence consistent with the peptide forming amyloid fibrils with a class 7 architecture. The results highlight the potential utility of phosphorylation as a method of reversibly controlling the aggregation kinetics of peptide sequences both during and after synthesis. Moreover, by exploiting the ability of the phosphate group to adopt different charge states as a function of pH, and combining experimental insights with atomistic information calculated from MD simulations as pH is varied, we show how the resulting information can be used to predict fibril structures consistent with both datasets, and use these to rationalise their sensitivity of fibrillation kinetics both to the location of the phosphate group and its charge state.

Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

PMID:
22174034
[PubMed - in process]
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9.
Biotechnol Appl Biochem. 2011 Nov;58(6):439-48. doi: 10.1002/bab.59. Epub 2011 Nov 3.

Conjugation to octa-arginine via disulfide bonds confers solubility to denatured proteins in physiological solution and enables efficient cell internalization.

Source

Medinet Medical Institute, MEDINET, Setagaya-ku, Tokyo, Japan.

Abstract

Some protein transduction methods have already been developed for regenerative medicine application. These methods can be applied to soluble proteins but not to insoluble proteins, such as those that originate from inclusion bodies, for example, Escherichia coli. We have developed a method that allows the in vitro solubilization of denatured proteins without refolding and their efficient cellular internalization through conjugation to the peptide, octa-arginine (R8), via disulfide bonds with cysteine residues. Ovalbumin (OVA), denatured in urea solution containing dithiothreitol, was used as a model protein. The R8 peptide was conjugated with OVA in urea solution. Denatured OVA was recovered in the insoluble fraction after dialysis against phosphate-buffered saline. However, almost all the R8-conjugated OVA was recovered in the soluble fraction and used for translocation experiments in HeLa, Chinese hamster ovary-K1, Cos-7, and matured dendritic cells, where efficient internalization of the protein conjugate was observed. Furthermore, we formulated R8-conjugated β-galactosidase and R8-conjugated luciferase using a similar procedure, and investigated how the conjugated proteins are processed after cell internalization. We also observed that only a small fraction of these proteins refolded and almost all underwent intracellular degradation. These results suggest that this method is suitable for the transduction of antigen-presenting cells and will benefit research and innovation in vaccine design and discovery.

Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.

PMID:
22172106
[PubMed - in process]
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10.
Methods Mol Biol. 2012;824:483-500.

A novel plant cell bioproduction platform for high-yield secretion of recombinant proteins.

Source

Arkansas Biosciences Institute, Arkansas State University, State University, AR, USA, jxu@astate.edu.

Abstract

Plant cell suspension culture integrates the merits of whole-plant systems with those of microbial fermentation and mammalian cell culture, and has been recognized as a promising alternative biosynthetic platform for valuable proteins. However, the low protein productivity dilemma has been the bottleneck toward commercializing this technology. Here, we describe a new technology, termed hydroxyproline (Hyp)-Glyco technology, that dramatically increases the yield of secreted recombinant proteins from cultured plant cells by expressing them as fusions with a novel glycomodule tag comprising an Hyp-rich repetitive peptide (HypRP) backbone that is subsequently glycosylated through the Hyp residues. The extensive glycosylation of the HypRP tags greatly extends the serum half-life of small therapeutic proteins, such as interferon α2b or human growth hormone, without significantly impairing their bioactivities and the tag greatly enhances solubility.

PMID:
22160916
[PubMed - in process]
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11.
Int J Biol Macromol. 2011 Nov 18. [Epub ahead of print]

Mechanistic insights into protein precipitation by alcohol.

Source

Institute of Applied Physics, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan.

Abstract

Ethanol is used to precipitate proteins during various processes, including purification and crystallization. To elucidate the mechanism of protein precipitation by alcohol, we have investigated the solubility and structural changes of protein over a wide range of alcohol concentrations. Conformation of hen egg-white lysozyme was changed from native to α-helical rich structure in the presence of ethanol at concentrations above 60%. The solubility of lysozyme was reduced with increasing ethanol concentration, although gel formation at ethanol concentrations between 60% and 75% prevented accurate solubility measurements. SH-modified lysozyme showed largely unfolded structure in water and α-helical structure in the presence of ethanol. More importantly, solubility of the chemically modified lysozyme molecules decreased with increasing ethanol concentration. There is no indication of increased solubility upon unfolding of the lysozyme molecules by ethanol, indicating that any favorable interaction of ethanol with the hydrophobic side chains, if indeed occuring, is offset by the unfavorable interaction of ethanol with the hydrophilic side chains and peptide bonds.

Copyright © 2011. Published by Elsevier B.V.

PMID:
22115717
[PubMed - as supplied by publisher]
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12.
AAPS J. 2011 Nov 22. [Epub ahead of print]

Absorption Enhancers: Applications and Advances.

Source

QPS, LLC, 110 Executive Drive, Suite 7, Newark, Delaware, 19702, USA, bruce.aungst@qps.com.

Abstract

Absorption enhancers are functional excipients included in formulations to improve the absorption of a pharmacologically active drug. The term absorption enhancer usually refers to an agent whose function is to increase absorption by enhancing membrane permeation, rather than increasing solubility, so such agents are sometimes more specifically termed permeation enhancers. Absorption enhancers have been investigated for at least two decades, particularly in efforts to develop non-injection formulations for peptides, proteins, and other pharmacologically active compounds that have poor membrane permeability. While at least one product utilizing an absorption enhancer for transdermal use has reached the market, quite a few more appear to be at the threshold of becoming products, and these include oral and transmucosal applications. This paper will review some of the most advanced absorption enhancers currently in development and the formulation technologies employed that have led to their success. In addition, a more basic review of the barriers to absorption and the mechanisms by which those barriers can be surmounted is presented. Factors influencing the success of absorption-enhancing formulations are discussed. If ultimately successful, the products now in development should offer non-injection alternatives for several peptide or protein drugs currently only administered by injection. The introduction of new absorption enhancers as accepted pharmaceutical excipients, and the development of formulation technologies that afford the greatest benefit/risk ratio for their use, may create opportunities to apply these enabling technologies more broadly to existing drugs with non-optimal delivery properties.

PMID:
22105442
[PubMed - as supplied by publisher]
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13.
Water Sci Technol. 2011;64(2):423-30.

Synthesis of polyaspartic acid-aminobenzenesulfonic acid grafted copolymer and its scale inhibition performance and dispersion capacity.

Source

School of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, China. hdccxu@126.com

Abstract

Polysuccinimide (abridged as PSI) was synthesized by urea and maleic anhydride. Aminobenzenesulfonic acid (ABSA) was introduced at different mole ratio to PSI to generate polyaspartic acid (abridged as PASP)/ABSA graft copolymer. The scale inhibition behavior of resultant PASP/ABSA copolymer was evaluated by using static scale inhibition method. The transmittance of the supernatant of the copolymer solution was measured to evaluate its dispersion ability for ferric oxide. The corrosion inhibition performance of the copolymer for iron plates immersed in the refined testing water (including 0.555 g of CaCl2 2H2O, 0.493 g of MgSO4 7H2O, 50 mg PASP/ABSA graft copolymer and 0.168 g of NaCl) was tested. It was found that PASP/ABSA copolymer was able to efficiently inhibit CaCO3 and Ca3(PO4)2 scales and had good corrosion inhibition ability as well, and it also had good dispersion ability for Fe2O3. Besides, the inhibition efficiency of PASP/ABSA against CaCO3 and Ca3(PO4)2 scales and its dispersion capacity for Fe2O3 was highly dependent on dosage. The reason may lie in that PASP/ABSA copolymer simultaneously possesses carboxylic ion and sulfonic group which can chelate Ca2+ to form stabilized and dissoluble chelates, resulting in increase of solubility of calcium salts in water. Also it may lie in that the introduction of acidic hydrophilic sulfonic group with a strong electrolytic capacity into PASP molecule simultaneously enhances the dispersion of the inhibitor molecules and hinders the formation of Ca3(PO4)2 scale.

PMID:
22097017
[PubMed - indexed for MEDLINE]
14.
Mol Neurodegener. 2011 Nov 17;6:77.

Superoxide dismutase 1 encoding mutations linked to ALS adopts a spectrum of misfolded states.

Source

Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL 32610, USA. borchelt@mbi.ufl.edu.

Abstract

ABSTRACT:

BACKGROUND:

Mutations in superoxide dismutase 1 (SOD1), which are one cause of familial amyotrophic lateral sclerosis (fALS), induce misfolding and aggregation of the protein. Misfolding can be detected by the binding of antibodies raised against peptide epitopes that are normally buried in the native conformation, shifts in solubility in non-ionic detergents, and the formation of macromolecular inclusions. In the present study, we investigate the relationship between detergent-insoluble and sedimentable forms of mutant SOD1, forms of mutant SOD1 with aberrantly accessible epitopes, and mutant protein in inclusions with the goal of defining the spectrum of misfolded states that mutant SOD1 can adopt.

RESULTS:

Using combined approaches in cultured cell models, we demonstrate that a substantial fraction of mutant SOD1 adopts a non-native conformation that remains soluble and freely mobile. We also show that mutant SOD1 can produce multimeric assemblies of which some are insoluble in detergent and large enough to sediment by ultracentrifugation and some are large enough to detect visually. Three conformationally restricted antibodies were found to be useful in discriminating mal-folded forms of mutant SOD1. An antibody termed C4F6 displays properties consistent with recognition of soluble, freely mobile, mal-folded mutant SOD1. An antibody termed SEDI, which recognizes C-terminal residues, detects larger inclusion structures as well as soluble misfolded entities. An antibody termed hSOD1, which recognizes aa 24-36, detects an epitope shared by soluble non-natively folded WT and mutant SOD1. This epitope becomes inaccessible in aggregates of mutant SOD1.

CONCLUSIONS:

Our studies demonstrate how different methods of detecting misfolding and aggregation of mutant SOD1 reveal different forms of aberrantly folded protein. Immunological and biochemical methods can be used in combination to detect soluble and insoluble misfolded forms of mutant SOD1. Our findings support the view that mutant SOD1 can adopt multiple misfolded conformations with the potential that different structural variants mediate different aspects of fALS.

PMID:
22094223
[PubMed - in process]
PMCID: PMC3248846
Free PMC Article
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15.
FEMS Microbiol Lett. 2011 Nov;324(2):98-105. doi: 10.1111/j.1574-6968.2011.02387.x. Epub 2011 Sep 15.

The pro-region of Streptomyces hygroscopicus transglutaminase affects its secretion by Escherichia coli.

Source

Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.

Abstract

Streptomyces transglutaminase (TGase) is secreted as a zymogen (pro-TGase) in liquid cultures and is then processed by the removal of its N-terminal region, resulting in active TGase. To date, there is no report describing TGase (or pro-TGase) secretion in Escherichia coli. In this study, the pro-TGase from Streptomyces hygroscopicus was efficiently secreted by E. coli BL21(DE3) using the TGase signal peptide or the pelB signal peptide. The secreted pro-TGase was efficiently transformed into active TGase by adding dispase to the culture supernatant of the recombinant strains. Mutational analysis showed that deletion of the first six amino acids of the N-terminal of the pro-region reduced the secretion of pro-TGase, and removal of the next 10 amino acids resulted in the formation of insoluble pro-TGase. These results suggest that the pro-region of TGase is essential for its efficient secretion and solubility in E. coli.

2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

PMID:
22092809
[PubMed - in process]
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16.
Expert Rev Proteomics. 2011 Dec;8(6):787-800.

Intact proteome fractionation strategies compatible with mass spectrometry.

Source

Department of Chemistry, Dalhousie University, 6274 Coburg Road, Halifax, NS, B3H 4R2, Canada. alan.doucette@dal.ca

Abstract

Proteome fractionation refers to separation at the level of intact proteins. Proteome fractionation may precede sample digestion and subsequent peptide-level separation and detection (i.e., bottom-up mass spectrometry [MS]). For top-down MS, proteome fractionation acts as a stand-alone separation platform, since intact proteins are directly analyzed by the mass spectrometer. Regardless of the MS identification strategy, separation of intact proteins has clear benefits as a result of decreasing sample complexity. However, this stage of the workflow also creates considerable challenges, which are generally absent from the counterpart peptide separation experiment. For example, maintaining proteinsolubility is a key concern before, during and after separation. To this end, surfactants such as sodium dodecyl sulfate may be employed during fractionation, so long as they are eliminated prior to MS. In this article, current strategies for proteome fractionation in a MS-compatible format are reviewed, illustrating the challenges and outlooks on this important aspect of proteomics.

© 2011 Expert Reviews Ltd

PMID:
22087661
[PubMed - in process]
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17.
Exp Biol Med (Maywood). 2011 Dec 1;236(12):1468-76. Epub 2011 Nov 15.

A new recombinant human apolipoprotein E mimetic peptide with high-density lipoprotein binding and function enhancing activity.

Source

Department of Cell Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, SC 29209, USA.

Abstract

We generated a novel human apolipoprotein E (apoE)-mimetic peptide, designated EpK. EpK contains an N-terminal cysteine residue, a low-density lipoprotein receptor-binding fragment, a 6 × lysine linker and a lipid-binding fragment. The recombinant peptide was expressed in Escherichia coli, and purified with a chitin bead column followed by a Heparin Sepharose CL-6B column to yield pure peptide. EpK displayed high solubility in aqueous solution at neutral pH and adopted a low content of α-helical structure which was significantly increased in 2,2,2-trifluoroethanol or upon lipid binding. EpK retained similar 1,2-dimyristoyl(d54)-sn-glycero-3-phosphocholine binding activity as human apoE3 albeit with slower kinetics. Cell culture studies showed that EpK mediated cholesterol efflux from cholesterol-loaded primary murine macrophages with higher mass-based efficiency than human apoAI and human apoE3, and that EpK inhibited lipopolysaccharide (LPS)-induced proinflammatory cytokine expression in murine macrophages. When injected into apoE(-/-)mice, EpK predominantly associated with high-density lipoprotein (HDL), which was also shown in in vitro incubation experiments. Moreover, association of EpK with HDL enhanced the ability of HDL in mediating cholesterol efflux and suppressing LPS-induced proinflammatory cytokine expression in cholesterol-loaded human acute monocytic leukemia cell line (THP-1) macrophages. These data suggest that this novel recombinant apoE mimetic peptideenhances HDL function and harbors antiatherogenic potential.

PMID:
22087021
[PubMed - in process]
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18.
J Sci Food Agric. 2011 Nov 14. doi: 10.1002/jsfa.4713. [Epub ahead of print]

Concentration-dependent displacement of cholesterol in micelles by hydrophobic rice bran protein hydrolysates.

Source

School of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China; US Department of Agriculture, Albany, CA 94710, USA.

Abstract

BACKGROUND:

Rice bran, containing about 100-150 g kg(-1) protein, is a by-product of rice milling that has only become an available ingredient in recent years owing to the centralisation of rice milling. Rice bran, but not its protein fraction or hydrolysates, has been shown to have a hypocholesterolaemic effect. Peptides from soy, milk and other foods have been proposed to have hypocholesterolaemic effects based on their ability to lower cholesterol solubility in bile acid/phosphotidyl choline micelles.

RESULTS:

Rice bran protein hydrolysates (RBPHs) were prepared and investigated for their potential to lower cholesterol concentration in micelles. The RBPHs were produced by digestion using four different peptidases, alcalase 2.4L(®) , neutrase 0.8L(®) , papaya latex papain and porcine pancreas trypsin, and then fractionated by hydrophobicity using styrene/divinylbeneze resins. Alcalase 2.4L(®) produced the highest degree of hydrolysis, and the resulting hydrolysates had the highest micellar cholesterol inhibition ability in an in vitro hypocholesterolaemic test. The adsorption dynamics of four different macroporous resins, DA201-C, Sepabeads SP207 and SP825 and Diaion HP20, were determined using the Langmuir isotherm model. DA201-C had the highest adsorption capacity with an equilibrium concentration of 220 mg g(-1) . The hydrolysates eluted with 25, 50, 75 and 95% (v/v) ethanol lowered the micellar cholesterol concentration by 11.88, 14.76, 19.37 and 7.56% respectively.

CONCLUSION:

A hydrophobic fraction of RBPH had the highest inhibitory activity on micellar cholesterol, which suggests that it may have hypocholesterolaemic properties. Copyright © 2011 Society of Chemical Industry.

Copyright © 2011 Society of Chemical Industry.

PMID:
22083811
[PubMed - as supplied by publisher]
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19.
Biochim Biophys Acta. 2012 Feb;1818(2):135-45. Epub 2011 Oct 28.

The role of membrane thickness in charged protein-lipid interactions.

Source

Department of Chemistry, University of California, Davis. One Shields Avenue, Davis, CA, 95616, USA.

Abstract

Charged amino acids are known to be important in controlling the actions of integral and peripheral membrane proteins and cell disrupting peptides. Atomistic molecular dynamics studies have shed much light on the mechanisms of membrane binding and translocation of charged protein groups, yet the impact of the full diversity of membrane physico-chemical properties and topologies has yet to be explored. Here we have performed a systematic study of an arginine (Arg) side chain analog moving across saturated phosphatidylcholine (PC) bilayers of variable hydrocarbon tail length from 10 to 18 carbons. For all bilayers we observe similar ion-induced defects, where Arg draws water molecules and lipid head groups into the bilayers to avoid large dehydration energy costs. The free energy profiles all exhibit sharp climbs with increasing penetration into the hydrocarbon core, with predictable shifts between bilayers of different thickness, leading to barrier reduction from 26kcal/mol for 18 carbons to 6kcal/mol for 10 carbons. For lipids of 10 and 12 carbons we observe narrow transmembrane pores and corresponding plateaus in the free energy profiles. Allowing for movements of the protein and side chain snorkeling, we argue that the energetic cost for burying Arg inside a thin bilayer will be small, consistent with recent experiments, also leading to a dramatic reduction in pK(a) shifts for Arg. We provide evidence that Arg translocation occurs via an ion-induced defect mechanism, except in thick bilayers (of at least 18 carbons) where solubility-diffusion becomes energetically favored. Our findings shed light on the mechanisms of ion movement through membranes of varying composition, with implications for a range of charged protein-lipid interactions and the actions of cell-perturbing peptides. This article is part of a Special Issue entitled: Membrane protein structure and function.

Copyright © 2011 Elsevier B.V. All rights reserved.

PMID:
22063722
[PubMed - in process]
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20.
Biochem Pharmacol. 2011 Oct 25. [Epub ahead of print]

Peptide therapeutics for CNS indications.

Source

PsychoGenics Inc., 765 Old Saw Mill River Road, Tarrytown, NY 10591, USA.

Abstract

Neuropeptides play a crucial role in the normal function of the central nervous system and peptide receptors hold great promise as therapeutic targets for the treatment of several CNS disorders. In general, the development of peptidetherapeutics has been limited by the lack of drug-like properties of peptides and this has made it very difficult to transform them into marketable therapeutic molecules. Some of these challenges include poor in vivo stability, poorsolubility, incompatibility with oral administration, shelf stability, cost of manufacture. Recent technical advances have overcome many of these limitations and have led to rapid growth in the development of peptides for a wide range of therapeutic indications such as diabetes, cancer and pain. This review examines the therapeutic potential of peptideagonists for the treatment of major CNS disorders such as schizophrenia, anxiety, depression and autism. Both clinical and preclinical data has been accumulated supporting the potential utility of agonists at central neurotensin, cholecystokinin, neuropeptide Y and oxytocin receptors. Some of the successful approaches that have been developed to increase the stability and longevity of peptides in vivo and improve their delivery are also described and potential strategies for overcoming the major challenge that is unique to CNS therapeutics, penetration of the blood-brain barrier, are discussed.

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