Assessment of new 6-Cl-HOBt based coupling reagents for peptide synthesis. Part 2: Racemization studies

Novel Cl-HOBt based coupling reagents have been evaluated forracemization extent in solid-phase peptide synthesis. The resultsshow that all the coupling protocols based on the use of the novel reagents enable incorporation of the racemization prone residue serine with less than 2% racemization. Moreover, serine racemization obtained is less than 0.5% with protocolswhere a pre-activation step is avoided.     

Assessment of new 6-Cl-HOBt based coupling reagents for peptide synthesis. Part 2: Racemization studies

Summary Novel Cl-HOBt based coupling reagents have been evaluated for racemization extent in solid-phase peptide synthesis. The results show that all the coupling protocols based on the use of the novel reagents enable incorporation of the racemization prone residue serine with less than 2% racemization. Moreover, serine racemization obtained is less than 0.5% with protocols where a pre-activation step is avoided.     

Assessment of new 6-Cl-HOBt based coupling reagents for peptide synthesis. Part 1: Coupling efficiency study

The efficiency of a series of well-known coupling reagents (TBTU, HATU, and PyBOP) and of new in situ activating reagents (TCTU, HCTU, and DMTMM) was compared by synthesizing the 65–74 fragment of the Acyl Carrier Protein (H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-NH₂₎, containing `a difficult sequence', as a test peptide, in a multiple peptide synthesizer. The longer sequence rMOG(35–55) was also synthesized. It was clear that the aminium salts are more efficient than the phosphonium salt (PyBOP) and that the new 6Cl-HOBt based reagents (HCTU and particularly TCTU) are very efficient, while DMTMM appeared to be not suitable for SPPS.     

Assessment of new 6-Cl-HOBt based coupling reagents for peptide synthesis. Part 1: Coupling efficiency study

Summary The efficiency of a series of well-known coupling reagents (TBTU, HATU, and PyBOP) and of newin situ activating reagents (TCTU, HCTU, and DMTMM) was compared by synthesizing the 65–74 fragment of the Acyl Carrier Protein (H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-NH₂), containing ‘a difficult sequence’, as a test peptide, in a multiple peptide synthesizer. The longer sequence rMOG(35–55) was also synthesized. It was clear that the aminium salts are more efficient than the phosphonium salt (PyBOP) and that the new 6Cl-HOBt based reagents (HCTU and particularly TCTU) are very efficient, while DMTMM appeared to be not suitable for SPPS.     

Bioprocess-centered molecular design (BMD) for the efficient production of an interfacially active peptide

The efficient expression and purification of an interfacially active peptide (mLac21) was achieved by using bioprocess-centered molecular design (BMD), wherein key bioprocess considerations are addressed during the initial molecular biology work. The 21 amino acid mLac21 peptide sequence is derived from the lac repressor protein and is shown to have high affinity for the oil-water interface, causing a substantial reduction in interfacial tension following adsorption. The DNA coding for the peptide sequence was cloned into a modified pET-31(b) vector to permit the expression of mLac21 as a fusion to ketosteroid isomerase (KSI). Rational iterative molecular design, taking into account the need for a scaleable bioprocess flowsheet, led to a simple and efficient bioprocess yielding mLac21 at 86% purity following ion exchange chromatography (and >98% following chromatographic polishing). This case study demonstrates that it is possible to produce acceptably pure peptide for potential commodity applications using common scaleable bioprocess unit operations. Moreover, it is shown that BMD is a powerful strategy that can be deployed to reduce bioseparation complexity.     

Discovery of highly potent novel antifungal azoles by structure-based rational design

On the basis of the active site of lanosterol 14α-demethylase from Candida albicans (CACYP51), a series of new azoles were designed and synthesized. All the new azoles show excellent in vitro activity against most of the tested pathogenic fungi, which represent a class of promising leads for the development of novel antifungal agents. The MIC₈₀ value of compounds 8c, 8i and 8n against C. albicans is 0.001 μg/mL, indicating that these compounds are more potent than fluconazole, itraconazole and voriconazole. Flexible molecular docking was used to analyze the structure–activity relationships (SARs) of the compounds. The designed compounds interact with CACYP51 through hydrophobic, van der Waals and hydrogen-bonding interactions.     

Stabilization and rational design of serine protease AprM under highly alkaline and high-temperature conditions.

Rational shift of the optimum pH toward alkalinity and enhancement of thermostability were investigated by using a thermostable extremely alkaline protease (optimum pH, 12 to 13) from the alkaliphilic and thermophilic Bacillus sp. strain B18'. The protease gene (aprM) was cloned, and the sequence analysis revealed an open reading frame of 361 amino acids that was composed of a putative signal sequence (24 amino acids), a prosequence (69 amino acids), and a mature enzyme (268 amino acids) (molecular weight, 27,664). The amino acid sequence of this protease was compared with those of other serine proteases. A direct correlation of higher optimum pH with an increase in the number of arginine residues was observed. An even more thermostable mutant enzyme was created by introducing a point mutation. When the position of the beta-turn, Thr-203, was replaced by Pro, the residual activity of this mutant enzyme at 80 degrees C for 30 min was higher than that of the wild-type enzyme (50% versus 10%). The specific activity of this mutant enzyme at 70 degrees C was 105% of that of the wild-type enzyme under nondenaturation condition. These data suggest that the higher content of Arg residues favors the alkalinity of the serine protease and that introduction of a Pro residue into the beta-turn structure stabilizes the enzyme.     

Non-destructive staging of barley reproductive development for molecular analysis based upon external morphology

A prerequisite to study the molecular genetic pathways of pollen and anther development is an accurate staging system for reproductive development. However in barley, floret formation occurs mainly within the pseudostem, which makes the observation of floret development and access to the floret particularly difficult without dissecting the plant. Thus selecting stages for molecular analysis cannot be done non-destructively. A staging method has therefore been developed for barley in order to define the relationship between readily detectable growth points and reproductive development, to provide a clear key to enable accurate selection of reproductive material. Initial staging followed the traditional Zadoks decimal system, with minor adaptations to stages 31-34 and stage 37. The later stages, from 37 onward, were replaced by growth staging based upon the last flag elongation (LFE) and the position occupied by the spike within the pseudostem. Spike size could be readily predicted by using the staging system incorporating Zadoks stages 31-37, supplemented with substages and by LFE staging to improve accuracy. The different spike sizes, as well as the LFE stages, showed a clear relationship to events occurring within the anther, as confirmed by light microscopy of the anthers. The defined relationship between spike size and development to anther development now makes possible the accurate prediction of anther and pollen progression by external staging. This, therefore, provides a mechanism for non-destructive selection of material for analysis that is critical for the molecular characterization of genes in anther and pollen development.     

Protein structure based rational design of ecdysone agonists

We review the impact of protein X-ray crystallography on the rational design of insecticides that act as agonists of the ligand-binding domain of the Ecdysone receptor (EcR). As the EcR is a target specific to insects, these compounds potentially constitute new chemical classes of safe insecticides. The increased insight relative to that from ligand-only based (Quantitative) Structure–Activity Relations (QSARs), classical 2D-Hansch type or 3D-CoMFA/CoMSIA (Comparative Molecular Field/Similarity Analysis), is discussed. The importance of protein X-ray structure determination in support of the discovery process is stressed as the simplistic lock-and-key picture fails due to the remarkable flexibility of the EcR ligand binding site. Several new non-steroidal chemical classes of ecdysone agonists, designed by guidance from protein X-ray studies, are described.     

The rational design of semisynthetic peroxidases

A semisynthetic peroxidase was designed by exploiting the structural similarity of the active sites of vanadium dependent haloperoxidases and acid phosphatases. Incorporation of vanadate ion into the active site of phytase (E.C. 3.1.3.8), which mediates in vivo the hydrolysis of phosphate esters, leads to the formation of a semisynthetic peroxidase, which catalyzes the enantioselective oxidation of prochiral sulfides with H(2)O(2) affording the S-sulfoxide, e.g. in 66% ee at 100% conversion for thioanisole. Under reaction conditions the semi-synthetic vanadium peroxidase is stable for over 3 days with only a slight decrease in turnover frequency. Polar water-miscible cosolvents, such as methanol, dioxane, and dimethoxyethane, can be used in concentrations of 30% (v/v) at a small penalty in activity and enantioselectivity. Among the transition metal oxoanions that are known to be potent inhibitors, only vanadate resulted in a semisynthetic peroxidase when incorporated into phytase. A number of other acid phosphatases and hydrolases were tested for peroxidase activity, when incorporated with vanadate ion. Phytases from Aspergillus ficuum, A. fumigatus, and A. nidulans, sulfatase from Helix pomatia, and phospholipase D from cabbage catalyzed enantioselective oxygen transfer reactions when incorporated with vanadium. However, phytase from A. ficuum was unique in also catalyzing the enantioselective sulfoxidation, albeit at a lower rate, in the absence of vanadate ion.     

A peptide mimetic of an anti-CD4 monoclonal antibody by rational design

The development of rational methods to design 'continuous' sequence mimetics of discontinuous regions of protein sequence has, to now, been only marginally successful. This has been largely due to the difficulty of constraining the recognition elements of a mimetic structure to the relative conformational and spatial orientations present in the parent molecule. Using peptide mapping to determine 'active' antigen recognition residues, molecular modeling, and a molecular dynamics trajectory analysis, we have developed a peptide mimic of an anti-CD4 antibody, containing antigen contact residues from multiple CDRs. The design described is a 27-residue peptide formed by juxtaposition of residues from 5 CDR regions. It displays an affinity for the antigen (CD4) of 0.9nM, compared to 2nM for the parent antibody ST40. Nevertheless, the mimetic shows low biological activity in an anti-retroviral assay.     

Terminal-modified polylysine-based chelating polymers: highly efficient coupling to antibody with minimal loss in immunoreactivity.

A method is suggested for the preparation of chelating polymers containing a single terminal reactive group capable of interaction with proteins. These polymers were synthesized from N-CBZ-polylysine and DTPA and contain a terminal SH or pyridyldisulfide group. A polymer molecule with MW 13,500 is able to carry up to 40 DTPA residues. Polymers easily and quantitatively bind with antibodies (Fab fragments of antimyosin antibodies R11D10) with minimal effect on antibody immunoreactivity as revealed in ELISA assay and in direct immunoanalysis. Conjugates prepared can chelate radioactive metal ions reaching very high specific radioactivity (greater than 1 mCi 111In/10 micrograms of protein). Perspectives for their application are discussed.     

A rational design of synthetic peptide vaccine with a built-in adjuvant. A modular approach for unambiguity.

We describe a peptide vaccine model containing a built-in adjuvant. This model used a multiple antigen peptide system (MAPS) to amplify peptide antigens and a lipoamino acid, tripalmitoyl glyceryl cysteine (P3C), as a built-in adjuvant. An 18-residue peptide antigen (B2) derived from the third variable domain (amino acid 312-329) of the glycoprotein gp120 of type I human immunodeficiency virus (HIV-1) was used in this model. This peptide antigen is a suitable target since it consists of neutralizing, T-helper, and T-cytotoxic epitopes. The peptide antigen in a tetravalent MAPS format (B2M-P3C) with a lipophilic attachment was synthesized by two routes for comparison: a direct stepwise approach and an indirect modular approach. In the stepwise approach, each residue was sequentially added to the peptide resin to give B2M-P3C and the P3C was incorporated to the side chain of a carboxyl terminal lysine as Fmoc-Lys(P3C). In the modular approach, a module containing a chloroacetylated core matrix of MAPS (M-P3C) with a carboxyl tetrapeptide bearing Lys(P3C) and a second module containing the peptide antigen B2 with a cysteine at its terminus were synthesized and purified separately, and then coupled to each other to form B2M-P3C. In the modular approach, the molecular ion of B2M-P3C was unambiguously identified by ion-spray mass spectrometry. B2M-P3C, administered in liposomes without any adjuvant such as Freund's complete adjuvant, was used to immunize mice and found to induce gp120-specific antibodies in vitro, and prime cytotoxic T lymphocytes in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exploitation of the selectivity-conferring code of nonribosomal peptide synthetases for the rational design of novel peptide antibiotics

Recently, the solved crystal structure of a phenylalanine-activating adenylation (A) domain enlightened the structural basis for the specific recognition of the cognate substrate amino acid in nonribosomal peptide synthetases (NRPSs). By adding sequence comparisons and homology modeling, we successfully used this information to decipher the selectivity-conferring code of NRPSs. Each codon combines the 10 amino residues of a NRPS A domain that are presumed to build up the substrate-binding pocket. In this study, the deciphered code was exploited for the first time to rationally alter the substrate specificity of whole NRPS modules in vitro and in vivo. First, the single-residue Lys239 of the L-Glu-activating initiation module C-A(Glu)-PCP of the surfactin synthetase A was mutated to Gln239 to achieve a perfect match to the postulated L-Gln-activating binding pocket. Biochemical characterization of the mutant protein C-A(Glu)-PCP(Lys239 --> Gln) revealed the postulated alteration in substrate specificity from L-Glu to L-Gln without decrease in catalytic efficiency. Second, according to the selectivity-conferring code, the binding pockets of L-Asp and L-Asn-activating A domains differs in three positions: Val299 versus Ile, His322 versus Glu, and Ile330 versus Val, respectively. Thus, the binding pocket of the recombinant A domain AspA, derived from the second module of the surfactin synthetases B, was stepwisely adapted for the recognition of L-Asn. Biochemical characterization of single, double, and triple mutants revealed that His322 represents a key position, whose mutation was sufficient to give rise to the intended selectivity-switch. Subsequently, the gene fragment encoding the single-mutant AspA(His322 --> Glu) was introduced back into the surfactin biosynthetic gene cluster. The resulting Bacillus subtilis strain was found to produce the expected so far unknown lipoheptapeptide [Asn(5)]surfactin. This indicates that site-directed mutagenesis, guided by the selectivity-conferring code of NRPS A domains, represents a powerful alternative for the genetic manipulation of NRPS biosynthetic templates and the rational design of novel peptide antibiotics.     

The relevance of cytokines for development of protective immunity and rational design of vaccines

Cytokines are key regulators of the immune system that shape innate and adaptive immune responses. An adequate balance of the cytokine environment is critical to achieve protective immunity and to avoid immunopathology. Present knowledge allows a deeper understanding of the cytokine network and their sometimes conflicting roles in the development of immune responses, as well as their relevance in the establishment and maintenance of immunological memory. New insights have been gained into the role of different T cell subsets for protection against infection or tumor growth. The incorporation of cytokines as molecular adjuvants in vaccines has been attempted to strengthen vaccine-induced immune responses, and as a rational approach to modulate cytokine milieu in vivo and tailor host immunity for specific situations. These approaches have been tried in experimental models and veterinary species, and a few of them have entered into clinical trials. However, manipulating the cytokine network to modulate immune responses is not a simple task, because cytokine functions are complex and the final effects on the immune response will depend on timing and length of exposure, cell(s) targeted and other cytokines present in the same microenvironment. Here, we will review our present understanding on the role of cytokines in the development of effector and memory T cell responses. Also the potential use of cytokines as molecular adjuvant for vaccines against infectious diseases and cancer will be revised.     

Solid phase peptide synthesis in water VI: evaluation of water-soluble coupling reagents for solid phase peptide synthesis in aqueous media

Solid phase peptide synthesis requires large amounts of organic solvents, the safe disposal of which is an important environmental issue. Peptide synthesis, if performed in water and using less or nontoxic reagents, circumvents the disposal problem. Our ultimate aim is to develop an "environment-friendly" solid phase peptide synthesis (SPPS) methodology. Previously, we showed that SPPS in water is feasible. To perform SPPS in water, the coupling reagent must be water-soluble and maintain its reactivity in water. For this report, we tested the efficacy of the water-soluble coupling reagents, 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium tetrafluoroborate (TNTU) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), towards SPPS in water. We successfully synthesized Leu-enkephalin amide on a solid support suspended in aqueous 50% EtOH using DMT-MM and 2-(4-sulfophenylsulfonyl)ethoxycarbonylamino acids.     

The rational design of an AIDS vaccine

The extraordinary genetic diversity and immune evasion of human immunodeficiency virus (HIV) pose significant challenges for vaccine development. AIDS vaccine design requires a scientifically driven, rational approach that encompasses the latest advances in viral molecular genetics, structural biology, and immunology.     

The design of antagonist peptide of hIL-6 based on the binding epitope of hIL-6 by computer-aided molecular modeling

The interaction between human interleukin-6 (hIL-6) and human interleukin-6 receptor (hIL-6R) is the initial and most specific step in the hIL-6 signaling pathway. Understanding its binding core and interaction mechanism at amino acid level is the basis for designing small IL-6 inhibiting molecules, such as peptides or lead compounds. With Docking method, the complex structure composed of hIL-6 and its alpha-subunit receptor (hIL-6R) was analyzed theoretically. By using structure-based analysis and phage display methods, the loop AB (from Lys67 to Glu81) of hIL-6 was found to be the important binding epitope of hIL-6R. By means of computer-aided design, the mimic antagonist peptide (14 residues) was designed and synthesized. Using multiple myeloma cell line (XG7), IL-6 dependent cell line, as test model, the influence of antagonist peptides on the proliferation of XG7 cells was investigated. The results showed that the synthetic peptide could be competitive to bind to hIL-6R with hIL-6, and the effect was concentration dependent. The theoretical design approach is a powerful alternative to phage peptide library for protein mimics. Such mini-peptide is more amenable to synthetic chemistry and thus may be useful starting points for the design of small organic mimics.     

The rational design of multiple molecular module-based assemblies for simultaneously improving rice yield and grain quality

正Rice is one of the most important staple food for over half of the world's population,and a substantial increase in productivity and quality of rice grain will be required to feed a growing human population.Grain size and shape are the two important components contributing to grain yield and quality,because they impact both yield potential and end-use quality.Over the past 50 years,the     

Fitting replacement of signal peptide for highly efficient expression of three penicillin G acylases in E. coli

Applied Microbiology and Biotechnology - High level expression of penicillin G acylase (PGA) in Escherichia coli is generally constricted by a complex maturation process and multiple limiting...