Cleavage Conditions Research Articles

Preparation of umami octopeptide with recombined Escherichia coli: Feasibility and challenges

ABSTRACTThe taste of umami peptide H-Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala-OH (LGAGGSLA) is controversial. One possible reason for this controversy is the use of chemically synthesized LGAGGSLA to confirm its taste. To explore other ways to further confirm the flavor of LGAGGSLA, we developed a new strategy to prepare a bio-source peptide by adopting a gene engineering method to express LGAGGSLA in recombinant Escherichia coli. In our previous work, we structured the LGAGGSLA recombinant expression system and optimized the culturing conditions for preparing a fusion protein. However, the fusion protein was not cleaved by enterokinase to obtain LGAGGSLA. Because the cleavage conditions of commercial enterokinase were not specific and recombinant engineered bacteria had the potential to be used in industrial processes, in this addendum, we calculated the mass and volume yields of key processing steps in the preparation of LGAGGSLA, and established a model of cleavage conditions with the cleavage ratio of LGAGGSLA. When the LGAGGSLA was confirmed to show umami taste, it is considered as a new umami or umami enhancer. The gene information of LGAGGSLA should have a great potential in the development of new flavor product and food product containing high umami flavor.

MgI2 -chemoselective cleavage for removal of amino acid protecting groups: A fresh vision for peptide synthesis.

In the field of peptide synthesis, the key to a successful access to synthetic targets lies on a pertinent combination of protecting groups. Their choice is directed by their selective removal conditions. We present here the behavior of some of the most used protecting groups in peptide chemistry under experimental cleavage conditions, combining MgI2 with MW irradiation, using 2-Me-THF as a green solvent. In these experimental conditions, the benzyloxycarbonyl protecting group as well as the Merrifield resin can be re-considered in peptide chemistry.

Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli

BackgroundAnimal venoms are large, complex libraries of bioactive, disulphide-rich peptides. These peptides, and their novel biological activities, are of increasing pharmacological and therapeutic importance. However, recombinant expression of venom peptides in Escherichia coli remains difficult due to the significant number of cysteine residues requiring effective post-translational processing. There is also an urgent need to develop high-throughput recombinant protocols applicable to the production of reticulated peptides to enable efficient screening of their drug potential. Here, a comprehensive study was developed to investigate how synthetic gene design, choice of fusion tag, compartment of expression, tag removal conditions and protease recognition site affect levels of solubility of oxidized venom peptides produced in E. coli.ResultsThe data revealed that expression of venom peptides imposes significant pressure on cysteine codon selection. DsbC was the best fusion tag for venom peptide expression, in particular when the fusion was directed to the bacterial periplasm. While the redox activity of DsbC was not essential to maximize expression of recombinant fusion proteins, redox activity did lead to higher levels of correctly folded target peptides. With the exception of proline, the canonical TEV protease recognition site tolerated all other residues at its C-terminus, confirming that no non-native residues, which might affect activity, need to be incorporated at the N-terminus of recombinant peptides for tag removal.ConclusionsThis study reveals that E. coli is a convenient heterologous host for the expression of soluble and functional venom peptides. Using the optimal construct design, a large and diverse range of animal venom peptides were produced in the µM scale. These results open up new possibilities for the high-throughput production of recombinant disulphide-rich peptides in E. coli.

IDENTIFICATION OF EPO-Fс FUSION PROTEIN BY MEANS OF POLYACRYLAMIDE GEL-ELECTROPHORESIS WITH ISOELECTROFOCUSING (IEF-PAGE)AND IN PRESENCE OF SODIUM DODECYLSULPHATE (SDS-PAGE)/ LAUROYLSARCOSINATE (SAR-PAGE) FOR THE PURPOSE OF ANTI-DOPING CONTROL

The article is devoted to develop of an approach for the identification of new stimulator of ematopoiesis, EPO-Fc fusion protein, which is banned by the World Anti-doping Agency (WADA) to use by athletes since it has become doping. Existing methods of qualitative determination of this substances in routine practice of antidoping laboratories such as polyacrylamide gelelectrophoresis in presence of sodium dodecylsulphate (SDS-PAGE) or lauroylsarcosinate (SARPAGE) are insufficiently specific. The article shows the principal possibility of identification of EPO-Fc fusion protein by means of IEF-PAGE in carrier ampholyte-based gels with a pH range 2-6 after Fc-fragment removal via fermentative hydrolysis.It has been shown that the removing of the crystallizable fragment leads to decrease of molecular weight of whole hybrid molecule and to increase its electrophoretic mobility that allows to detect this banned substances with high specificity by existing methods. During the study the enzyme for hydrolytic cleavage and optimum conditions of hydrolysis of EPO-Fc in serum samples were selected.

HF-Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization.

We have developed a convenient method for the direct synthesis of peptide thioesters, versatile intermediates for peptide ligation and cyclic peptide synthesis. The technology uses a modified Boc SPPS strategy that avoids the use of anhydrous HF. Boc in situ neutralization protocols are used in combination with Merrifield hydroxymethyl resin and TFA/TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS to post‐translational modifications that are compatible with the milder cleavage conditions, demonstrated here with the synthesis of the phosphorylated protein CHK2. Peptide thioesters give easy, direct, access to cyclic peptides, illustrated by the synthesis of cyclorasin, a KRAS inhibitor.

HF‐Free Boc Synthesis of Peptide Thioesters for Ligation and Cyclization

AbstractWe have developed a convenient method for the direct synthesis of peptide thioesters, versatile intermediates for peptide ligation and cyclic peptide synthesis. The technology uses a modified Boc SPPS strategy that avoids the use of anhydrous HF. Boc in situ neutralization protocols are used in combination with Merrifield hydroxymethyl resin and TFA/TMSBr cleavage. Avoiding HF extends the scope of Boc SPPS to post‐translational modifications that are compatible with the milder cleavage conditions, demonstrated here with the synthesis of the phosphorylated protein CHK2. Peptide thioesters give easy, direct, access to cyclic peptides, illustrated by the synthesis of cyclorasin, a KRAS inhibitor.

Effects of Electrical Pulse and 6-DMAP on Cleavage of Golden Hamster Oocytes—Morphological and Phisiological Observations

The golden hamster is a well-established model system for studies of morphology, reproductive physiology, oncology, genetics and virology. The aim of this study was to establish experimental protocols necessary for cloning the golden hamster; we examined and optimized conditions for parthenogenesis and cleavage of its oocytes. We tested oocytes of different ages, including 15 h after Human chorionic gonadotropin (hCG), with two treatments: (1) an electrical pulse ranging from 10 to 600 V/mm and (2) incubation for 2 to 6 h in 2 mM 6-dimethylaminopurine (6-DMAP). These two conditions were tested both separately and in combination. We found that (i) in oocytes of different ages, cleavage exhibits a strength-dependent increase; (ii) 6-DMAP stimulates oocyte cleavage, but the cleavage rates are significantly low; and (iii) a combined treatment is more effective than a treatment with 6-DMAP alone, and is comparable to those achieved with high pulse stimuli. These results elucidate certain parameters important for the cloning of the golden hamster species.

The Use of α-Halo Ethers in the Protection of Alcohols. Years 2012 - 2014

α-Halo ethers used in the protection of alcohols for the years 2012 - 2014 have been reviewed. The conditions for formation and cleavage of protected alcohols are presented in tables. The α-halo ethers that have been reviewed are: benzyloxymethyl chloride (BOMCl), ethoxymethyl chloride (EOMCl), 2-methoxyethoxymethyl chloride (MEMCl), methoxymethyl bromide (MOMBr), methoxymethyl chloride (MOMCl) and 2-(trimethylsilyl)ethoxymethyl chloride (SEMCl). Keywords: Acetals, protecting reagents, protection of alcohols, α-Halo ethers.

Selective functionalization of the primary hydroxy group in triols

A procedure was suggested for preparing functionally substituted 1,2-diols by O-alkylation of hydroxyalkyl-1,3-dioxacyclanes with alkyl halides, followed by acid hydrolysis. The conditions for selective cleavage of the ethers obtained were found. (2,2-Dimethyl-1,3-dioxolan-4-yl)methyl benzoate under the conditions of acid deacetalization undergoes saponification of the ester group.

Aromatic monomers by in situ conversion of reactive intermediates in the acid-catalyzed depolymerization of lignin.

Conversion of lignin into well-defined aromatic chemicals is a highly attractive goal but is often hampered by recondensation of the formed fragments, especially in acidolysis. Here, we describe new strategies that markedly suppress such undesired pathways to result in diverse aromatic compounds previously not systematically targeted from lignin. Model studies established that a catalytic amount of triflic acid is very effective in cleaving the β-O-4 linkage, most abundant in lignin. An aldehyde product was identified as the main cause of side reactions under cleavage conditions. Capturing this unstable compound by reaction with diols and by in situ catalytic hydrogenation or decarbonylation lead to three distinct groups of aromatic compounds in high yields acetals, ethanol and ethyl aromatics, and methyl aromatics. Notably, the same product groups were obtained when these approaches were successfully extended to lignin. In addition, the formation of higher molecular weight side products was markedly suppressed, indicating that the aldehyde intermediates play a significant role in these processes. The described strategy has the potential to be generally applicable for the production of interesting aromatic compounds from lignin.

Deciphering EGFP production via surface display and self-cleavage intein system in different hosts

In this study, six E. coli strains, which harboring enhanced green fluorescence protein (EGFP), an intein (INT) and ice nucleation protein (INP), were used to express the EGFP protein. The effect of E. coli strains on the surfaced displayed EGFP via INP-INT system was first investigated. Among these strains, E. coli JM109(DE3) was found the best strain for EGFP production (67.9 mg/L), two-fold as compared to that produced by E. coli DH1(DE3). It was concluded that lon mutation could stabilize recombinant proteins. Thus, the deletion of the gene coding for proteolysis might be a critical factor when using host for surface-expression production. The optimal conditions for cell cultivation, gene induction and intein cleavage for EGFP production were systematically studied and discussed. The objective function (O) value of 26.5 can be reached at a 12-h induction and 20-h cleavage process, where the EGFP yield is 58.6 mg/L with a purity of 45.3%. Furthermore, results indicated that the higher intein activity was obtained in the shorter cleavage duration; however, the shorter cleavage time sacrificed the production yield as expected. To compromise between the production yield and purity, the optimal conditions for the EGFP production were: 12-h induction and 20-h cleavage process is for the production of higher purity EGFP.

Quantitative analysis of PMLA nanoconjugate components after backbone cleavage.

Multifunctional polymer nanoconjugates containing multiple components show great promise in cancer therapy, but in most cases complete analysis of each component is difficult. Polymalic acid (PMLA) based nanoconjugates have demonstrated successful brain and breast cancer treatment. They consist of multiple components including targeting antibodies, Morpholino antisense oligonucleotides (AONs), and endosome escape moieties. The component analysis of PMLA nanoconjugates is extremely difficult using conventional spectrometry and HPLC method. Taking advantage of the nature of polyester of PMLA, which can be cleaved by ammonium hydroxide, we describe a method to analyze the content of antibody and AON within nanoconjugates simultaneously using SEC-HPLC by selectively cleaving the PMLA backbone. The selected cleavage conditions only degrade PMLA without affecting the integrity and biological activity of the antibody. Although the amount of antibody could also be determined using the bicinchoninic acid (BCA) method, our selective cleavage method gives more reliable results and is more powerful. Our approach provides a new direction for the component analysis of polymer nanoconjugates and nanoparticles.

Regioselective solid-phase synthesis of N-mono-hydroxylated and N-mono-methylated acylpolyamine spider toxins using an 2-(ortho-nitrophenyl)ethanal-modified resin.

A recently introduced new SPS resin, possessing a 2-(ortho-nitrophenyl)ethanal linker, was used for the regioselective on-resin synthesis of N-mono-hydroxylated and N-mono-methylated polyamine spider toxins of Agelenopsis aperta and Larinioides folium. The polyamine backbones of the target compounds were efficiently constructed from the center by reductive amination of the aldehyde linker, followed by stepwise alkylation and acylation on solid support. Depending on the cleavage conditions, employing either oxidation/Cope elimination or methylation/Hofmann elimination, regioselectively the respective N-hydroxyl or N-methyl products were obtained. Employing this methodology, a number of acylpolyamine spider toxins were synthesized and identified as venom components by UHPLC and ESI-MS/MS.

Optimization of an Indirect Enzymatic Method for the Simultaneous Analysis of 3-MCPD, 2-MCPD, and Glycidyl Esters in Edible Oils.

We developed a novel, indirect enzymatic method for the analysis of fatty acid esters of 3-monochloro-1,2-propanediol (3-MCPD), 2-monochloro-1,3-propanediol (2-MCPD), and glycidol (Gly) in edible oils and fats. Using this method, the ester analytes were rapidly cleavaged by Candida rugosa lipase at room temperature for 0.5 h. As a result of the simultaneous hydrolysis and bromination steps, 3-MCPD esters, 2-MCPD esters, and glycidyl esters were converted to free 3-MCPD, 2-MCPD, and 3-monobromo-1,2-propanediol (3-MBPD), respectively. After the addition of internal standards, the mixtures were washed with hexane, derivatized with phenylboronic acid, and analyzed by gas chromatography-mass spectrometer (GC-MS). The analytical method was evaluated in preliminary and feasibility studies performed by 13 laboratories. The preliminary study from 4 laboratories showed the reproducibility (RSD R ) of < 10% and recoveries in the range of 102-111% for the spiked 3-MCPD and 2-MCPD in extra virgin olive (EVO) oil, semi-solid palm oil, and solid palm oil. However, the RSDR and recoveries of Gly in the palm oil samples were not satisfactory. The Gly content of refrigerated palm oil samples decreased whereas the samples at room temperature were stable for three months, and this may be due to the depletion of Gly during cold storage. The feasibility studies performed by all 13 laboratories were conducted based on modifications of the shaking conditions for ester cleavage, the conditions of Gly bromination, and the removal of gel formed by residual lipase. Satisfactory RSDR were obtained for EVO oil samples spiked with standard esters (4.4% for 3-MCPD, 11.2% for 2-MCPD, and 6.6% for Gly).

Synthesis of anionic peptide nucleic acid oligomers including γ-carboxyethyl thymine monomers

Synthesis of a modified thymine monomer based on l-Glu, solid phase synthesis of anionic peptide nucleic acid (PNA) oligomers containing the modified monomer and optimization of the deblocking procedure and cleavage conditions of the PNA oligomers from resin are accomplished.

Phosphoryl Guanidines: A New Type of Nucleic Acid Analogues

A new type of nucleic acid analogues with a phosphoryl guanidine group is described. Oxidation of polymer-supported dinucleoside 2-cyanoethyl phosphite by iodine in the presence of 1,1,3,3-tetramethyl guanidine yields a dinucleotide with an internucleoside tetramethyl phosphoryl guanidine (Tmg) group as the main product. The Tmg group is stable under conditions of solid-phase DNA synthesis and subsequent cleavage and deprotection with ammonia. Oligonucleotides with one or more Tmg groups bind their complementary DNA or RNA with affinity similar to that of natural oligodeoxyribonucleotides.

Design and identification of a high efficient formic acid cleavage site for separation of fusion protein.

The release of target protein with high efficiency and low cost from expressed fusion protein is a key requirement for commercial production of target proteins. To establish such a cleavage system, we have designed four formic acid (FA) cleavage sites C1 (DPDPDP), C2 (DPPDPP), C3 (DDDDPI) and C4 (IVDPNP), which was placed in between the E and G fusion protein. Four expression vectors were individually constructed and expressed in Escherichia coli. Purified proteins were reacted with a series of FA concentrations or under different temperatures followed by SDS-PAGE gel electrophoresis to verify the degree of cleavage efficiency. Results showed that the C2 was the most efficient site compared with the other three. After optimization of cleavage conditions for E-C2-G, the cleavage efficiently could reach as high as 87.3% within 2.5 h in 37% FA at 45 °C. Comparing with previous reports, a significant reduction (26%) of FA concentration at a lower temperature in a short duration of reaction (18 times less) was achieved. We believe the cleavage site of DPPDPP identified in this study can be used in the large-scale production of valuable fusion proteins to save the cost, time and energy.

DNA methylation reduces binding and cleavage by bleomycin.

In a recent study, we described the enhanced double-strand cleavage of hairpin DNAs by Fe·bleomycin (Fe·BLM) that accompanies increasingly strong binding of this antitumor agent and suggested that this effect may be relevant to the mechanism by which BLM mediates its antitumor effects. Because the DNA in tumor cells is known to be hypomethylated on cytidine relative to that in normal cells, it seemed of interest to study the possible effects of methylation status on BLM-induced double-strand DNA cleavage. Three hairpin DNAs found to bind strongly to bleomycin, and their methylated counterparts, were used to study the effect of methylation on bleomycin-induced DNA degradation. Under conditions of limited DNA cleavage, there was a significant overall decrease in the cleavage of methylated hairpin DNAs. Cytidine methylation was found to result in decreased BLM-induced cleavage at the site of methylation and to result in enhanced cleavage at adjacent nonmethylated sites. For two of the three hairpin DNAs studied, methylation was accompanied by a dramatic decrease in the binding affinity for Fe·BLM, suggesting the likelihood of diminished double-strand cleavage. The source of the persistent binding of BLM by the third hairpin DNA was identified. Also identified was the probable molecular mechanism for diminished binding and cleavage of the methylated DNAs by BLM. The possible implications of these findings for the antitumor selectivity of bleomycin are discussed.

N-O Cleavage reactions of heterobicycloalkene-fused 2-isoxazolines.

Transition metal-mediated N–O bond cleavage reactions of heterobicycloalkene-fused 3-methyl-2-isoxazolines were investigated. Optimal cleavage conditions were found with Raney nickel/AlCl3 mediation in aqueous methanol. The reaction provided a diverse collection of novel heterobicycle-fused β-hydroxyketones with good to excellent yields (66–95%) and without the need for chromatographic purification.

A New Method for Synthesis of Peptide Thioesters via Irreversible N-to-S Acyl Transfer

A new synthetic method for peptide thioesters is described using Fmoc solid-phase peptide synthesis (Fmoc-SPPS). This method employs a novel enamide motif to facilitate irreversible intramolecular N-to-S acyl migration, which can efficiently afford the desired peptide thioesters (3 h, 30 °C) under the final trifluoroacetic acid (TFA) cleavage conditions. The acyl-transfer-mediated approach for synthesis of peptide thioesters tolerated different C-terminal residues and was used to synthesize human C-C motif chemokine 11 (hCCL11) via native chemical ligation.