Importance of expression system in the production of unnatural recombinant proteins in Escherichia coli

In this study, we investigated the efficiencies by which the pET and pQE expression systems produce unnatural recombinant proteins by residue-specific incorporation of unnatural amino acids, a method through which it was found that type of gene expression system tremendously influences the production yield of unnatural proteins in Escherichia coli. Green fluorescent protein (GFP) and a single-chain Fv antibody against c-Met were utilized as model recombinant proteins while L-homopropargylglycine (Hpg), a methionine analogue that incorporates into the methionine residues of a recombinant protein, was used as model unnatural amino acid. The pET system produced an almost negligible amount of Hpg-incorporated unnatural protein compared to the amount of methionine-incorporated natural protein. However, comparable amounts of unnatural and natural protein were produced by the pQE expression system. The amount of unnatural GFP protein produced through pET expression was not increased despite the over-expression of methionyl tRNA synthetase, which can enhance the activation rate of methionyl-tRNA with a methionine analogue. Incorporation of Hpg decreased the productivity of active GFP by approximately 2.5 fold, possibly caused by the inefficient folding of Hpg-incorporated GFP. Conversely, the productivity of functional anti-c-Met sc-Fv was not influenced by incorporation of Hpg. We confirmed through LC-MS and LCMS/MS that Hpg was incorporated into the methionine residues of the recombinant proteins produced by the pQE expression system. The first two authors equally contributed to this work.     

First asymmetric synthesis of (-)-lintetralin via intramolecular Friedel-Crafts-type cyclization

The asymmetric synthesis of an aryltetralin lignan, (-)-lintetralin, was achieved with an overall yield of 29% with seven steps. Key features of the synthesis are an asymmetric Strecker reaction, a diastereoselective Michael addition of the lithiated amino nitrile product to 5H-furan-2-one, and an intramolecular carbocationic cyclization to provide the desired ring skeleton with the correct configuration.     

Effects of arginine and leucine substitutions on anti‐endotoxic activities and mechanisms of action of cationic and amphipathic antimicrobial octadecapeptide from rice α‐amylase

Previously, we showed that the antimicrobial cationic and amphipathic octadecapeptide AmyI‐1‐18 from rice α‐amylase (AmyI‐1) inhibited the endotoxic activity of lipopolysaccharide (LPS) from Escherichia coli. In addition, we demonstrated that several AmyI‐1‐18 analogs containing arginine or leucine substitutions, which were designed on the basis of the helical wheel projection of AmyI‐1‐18, exhibited higher antimicrobial activity against human pathogenic microorganisms than AmyI‐1‐18. In the present study, anti‐inflammatory (anti‐endotoxic) activities of five AmyI‐1‐18 analogs containing arginine or leucine substitutions were investigated. Two single arginine‐substituted and two single leucine‐substituted AmyI‐1‐18 analogs inhibited the production of LPS‐induced nitric oxide in mouse macrophages (RAW264) more effectively than AmyI‐1‐18. These data indicate that enhanced cationic and hydrophobic properties of AmyI‐1‐18 are associated with improved anti‐endotoxic activity. In subsequent chromogenic Limulus amebocyte lysate assays, 50% inhibitory concentrations (IC₅₀) of the three AmyI‐1‐18 analogs (G12R, D15R, and E9L) were 0.11–0.13 μm , indicating higher anti‐endotoxic activity than that of AmyI‐1‐18 (IC_50, 0.22 μm ), and specific LPS binding activity. In agreement, surface plasmon resonance analyses confirmed direct LPS binding of three AmyI‐1‐18 analogs. In addition, AmyI‐1‐18 analogs exhibited little or no cytotoxic activity against RAW264 cells, indicating that enhancements of anti‐inflammatory and LPS‐neutralizing activities following replacement of arginine or leucine did not result in significant increases in cytotoxicity. This study shows that the arginine‐substituted and leucine‐substituted AmyI‐1‐18 analogs with improved anti‐endotoxic and antimicrobial activities have clinical potential as dual‐function host defense agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Structural investigation on WlaRG from Campylobacter jejuni: A sugar aminotransferase

Campylobacter jejuni is a Gram‐negative bacterium that represents a leading cause of human gastroenteritis worldwide. Of particular concern is the link between C. jejuni infections and the subsequent development of Guillain‐Barré syndrome, an acquired autoimmune disorder leading to paralysis. All Gram‐negative bacteria contain complex glycoconjugates anchored to their outer membranes, but in most strains of C. jejuni, this lipoglycan lacks the O‐antigen repeating units. Recent mass spectrometry analyses indicate that the C. jejuni 81116 (Penner serotype HS:6) lipoglycan contains two dideoxyhexosamine residues, and enzymological assay data show that this bacterial strain can synthesize both dTDP‐3‐acetamido‐3,6‐dideoxy‐d ‐glucose and dTDP‐3‐acetamido‐3,6‐dideoxy‐d ‐galactose. The focus of this investigation is on WlaRG from C. jejuni, which plays a key role in the production of these unusual sugars by functioning as a pyridoxal 5′‐phosphate dependent aminotransferase. Here, we describe the first three‐dimensional structures of the enzyme in various complexes determined to resolutions of 1.7 Å or higher. Of particular significance are the external aldimine structures of WlaRG solved in the presence of either dTDP‐3‐amino‐3,6‐dideoxy‐d ‐galactose or dTDP‐3‐amino‐3,6‐dideoxy‐d ‐glucose. These models highlight the manner in which WlaRG can accommodate sugars with differing stereochemistries about their C‐4′ carbon positions. In addition, we present a corrected structure of WbpE, a related sugar aminotransferase from Pseudomonas aeruginosa, solved to 1.3 Å resolution.     

An improved synthesis of (2S, 4S)‐ and (2S, 4R)‐2‐amino‐4‐methyldecanoic acids: assignment of the stereochemistry of culicinins

An improved synthesis of (2S, 4S)‐ and (2S, 4R)‐2‐amino‐4‐methyldecanoic acids was accomplished using a glutamate derivative as starting material and Evans' asymmetric alkylation as the decisive step. The NMR data of the two diastereomers were measured and compared with those of the natural product. As a result, the stereochemistry of this novel amino acid unit in culicinins was assigned as (2S, 4R). Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Evaluation of chiral separation efficiency of a novel OTPTHE derivatization reagent: Applications to liquid‐chromatographic determination of DL‐serine in human plasma

A novel chiral derivatization reagent, the N‐[1‐oxo‐5‐(triphenylphosphonium)pentyl]‐ (R)‐1,3‐thiazolidinyl‐4‐N‐hydroxysuccinimide ester bromide salt (OTPTHE), was developed for the separation and selective detection of chiral DL‐amino acids by RP‐HPLC analysis. The OTPTHE reacted with DL‐amino acids at 60°C maintained for 30 minutes in the presence of 100 mM borate buffer (pH 9.5). The separability of the diastereomeric derivatives was evaluated in terms of the resolution value (Rs) using 13 kinds of DL‐amino acids, which were completely separated by reversed‐phase chromatography using C18 column at 254 nm. The Rs of the DL‐amino acids varied from 1.62 to 2.51. As for the application of the DL‐amino acids, the determination of DL‐Ser in the human plasma of healthy volunteers was performed based on our developed method. It was shown that linear calibrations were available with high coefficients of correlation (r² > 0.9997). The limit of detection (S/N = 3) of the DL‐Ser enantiomers was 5.0 pmol; the relative standard deviations of the intraday and interday variations were below 4.56%; the accuracy ranged between 95.40%‐110.06% and 95.45%‐109.80%, respectively; the mean recoveries (%) of the DL‐Ser spiked in the human plasma were 99.49%‐103.74%. The amounts of DL‐Ser in the human plasma of healthy volunteers were determined.     

Preparation and in vitro antioxidant activities of 6‐amino‐6‐deoxychitosan and its sulfonated derivatives

The 6‐amino‐6‐deoxychitosan (NC) and their 2, 6‐di‐N‐sulfonated derivatives were prepared via N‐phthaloylation, tosylation, azidation, hydrazinolysis, reduction of azide groups and N‐sulfonation, and their structures were systematically characterized by FT‐IR, 2D HSQC NMR, XRD, gel permeation chromatography (GPC), and elemental analysis. The 6‐amino‐6‐deoxychitosan showed effect in three selected antioxidant essays, including reducing power, superoxide anion radical scavenging ability, and hydroxyl radical scavenging effect. But the factors affecting each activity were different. The reducing power and the superoxide anion radical scavenging ability of NC were strong and closely related to the amino groups in the molecular chains. Both introducing N‐sulfonated groups into NC and the concentration reduction of NC and its sulfonated derivatives decreased these activities. For the superoxide anion radical, the molecular charge property was also a significant influence factor. For the hydroxyl radical, NC only showed weak scavenging activity in a special inverse concentration‐dependent manner. However, the incorporation of N‐sulfonated groups significantly improved the scavenging activity, and the more N‐sulfonated groups, the higher the concentrations, the stronger the activity was. The results could be due to the different conformations of NC and its sulfonated derivatives in aqueous solution. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 539–549, 2015.     

Two-color labeling of temporally defined protein populations in mammalian cells

The proteome undergoes complex changes in response to disease, drug treatment, and normal cellular signaling processes. Characterization of such changes requires methods for time-resolved protein identification and imaging. Here, we describe the application of two reactive methionine (Met) analogues, azidohomoalanine (Aha) and homopropargylglycine (Hpg), to label two protein populations in fixed cells. Reactive lissamine rhodamine (LR), 7-dimethylaminocoumarin (DMAC), and bodipy-630 (BDPY) dyes were prepared and examined for use in selective dye-labeling of newly synthesized proteins in Rat-1 fibroblasts. The LR and DMAC, but not BDPY, fluorophores were found to enable selective, efficient labeling of subsets of the proteome; cells labeled with Aha and Hpg exhibited fluorescence emission three- to sevenfold more intense than that of control cells treated with Met. We also examined simultaneous and sequential pulse-labeling of cells with Aha and Hpg. After pulse-labeling, cells were treated with reactive LR and DMAC dyes, and labeled cells were imaged by fluorescence microscopy and analyzed by flow cytometry. The results of these studies demonstrate that amino acid labeling can be used to achieve selective two-color imaging of temporally defined protein populations in mammalian cells.     

A practical diastereoselective synthesis of (−)‐bestatin

Diastereoselective addition of nitromethane to Boc‐D‐Phe‐H in the presence of sodium hydride in diethyl ether/hexane containing 15‐crown‐5 and subsequent N,O‐protection with 2,2‐dimethoxypropane gave trans‐oxazolidine in a diastereomeric ratio of >16:1. The oxazolidine was easily separated by column chromatography, which after Nef reaction was coupled to H‐Leu‐OtBu. The 8‐step synthesis afforded (?)‐bestatin in an overall yield of 24.7% after deprotection and ion exchange.     

Efficient one‐pot synthesis of N‐ethyl amino acids

Mono‐N‐ethylated α‐amino acid esters are obtained in high yields using reductive amination procedures. Formation of imine is achieved by excess of acetaldehyde, followed by removal of acetaldehyde and reduction by NaBH(OAc)₃. The elaborated one‐pot synthesis allows for the efficient synthesis of side‐chain protected amino acid derivatives. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Highly sensitive and selective electrochemiluminescence determination of cholesterol utilizing a functional electrode with a core–shell nanostructure

A highly sensitive and selective method for the determination of cholesterol is required to evaluate trace amounts of cholesterol in test samples. In this work, selected gold nanoparticles (AuNPs) and 5‐amino‐2‐mercapto‐1,3,4‐thiadiazole (AMT) were used and a thin film of three‐dimensional gold–AMT core–shell nanoparticles (p‐AMT–AuNPs) was prepared using an electrochemical method. Cholesterol oxidase was then bonded to the film surface to give a functional electrode. Based on catalysis by the electrode functionalized for cholesterol and a luminol–H₂O₂ electrochemiluminescence (ECL) system, a highly sensitive and selective ECL method was developed for the determination of cholesterol. Under optimized conditions, ECL intensity showed a good linear relationship with cholesterol over the concentration range 0.05–11.0 µg/ml, with a correlation coefficient of 0.999 and a limit of detection of 0.02 µg/ml. The proposed method was used to determine cholesterol in dairy products with a relative standard deviation of < 1.8% and recovery rates of 98.1–104%. Copyright © 2015 John Wiley & Sons, Ltd.     

Overexpression of GmAAP6a enhances tolerance to low nitrogen and improves seed nitrogen status by optimizing amino acid partitioning in soybean

Amino acid transport via phloem is one of the major source‐to‐sink nitrogen translocation pathways in most plant species. Amino acid permeases (AAPs) play essential roles in amino acid transport between plant cells and subsequent phloem or seed loading. In this study, a soybean AAP gene, annotated as GmAAP6a, was cloned and demonstrated to be significantly induced by nitrogen starvation. Histochemical staining of GmAAP6a:GmAAP6a‐GUS transgenic soybean revealed that GmAAP6a is predominantly expressed in phloem and xylem parenchyma cells. Growth and transport studies using toxic amino acid analogs or single amino acids as a sole nitrogen source suggest that GmAAP6a can selectively absorb and transport neutral and acidic amino acids. Overexpression of GmAAP6a in Arabidopsis and soybean resulted in elevated tolerance to nitrogen limitation. Furthermore, the source‐to‐sink transfer of amino acids in the transgenic soybean was markedly improved under low nitrogen conditions. At the vegetative stage, GmAAP6a‐overexpressing soybean showed significantly increased nitrogen export from source cotyledons and simultaneously enhanced nitrogen import into sink primary leaves. At the reproductive stage, nitrogen import into seeds was greatly enhanced under both sufficient and limited nitrogen conditions. Collectively, our results imply that overexpression of GmAAP6a enhances nitrogen stress tolerance and source‐to‐sink transport and improves seed quality in soybean. Co‐expression of GmAAP6a with genes specialized in source nitrogen recycling and seed loading may represent an interesting application potential in breeding.     

Effects of indole‐3‐butyric acid on growth,pigments and UV‐screening compounds in Nostoc linckia

In the present research, the effect of indole‐3‐butyric acid (IBA) on the growth, and the production of some primary and secondary metabolites was studied in Nostoc linckia. In this respect, algae cultures were supplied with 0, 0.01, 0.1, 1, 10, and 100 μM IBA for 14 days. IBA at concentrations of 10 and 100 μM induced algal growth expressed as fresh weight in N. linckia. Treatment with IBA at all concentrations stimulated heterocyst formation. In addition, low concentrations of IBA (0.01, 0.1, and 1 μM) had a stimulatory effect on chlorophyll a and carotenoids accumulation. In contrast, higher concentrations of IBA induced the accumulation of phycocyanin, allophycocyanin, and phycoerythrin in the treated algae. In this case, IBA at the concentration of 10 μM was more effective. A significant decrease in protein content was observed in the algae treated by 0.01 μM IBA. All concentrations of IBA caused a decrease in sugar content, but lower concentrations were more effective. IBA application in all of the concentrations except 100 μM increased oligosaccharide‐linked mycosporine‐like amino acids (OS‐MAAs) content. Lower concentrations had a more significant effect on increasing OS‐MAAs content. However the concentrations of 10 and 100 μM IBA decreased scytonemin content. These results indicated the stimulatory impact of IBA on weight, heterocyst formation, and photosynthetic pigments in N. linckia.     

An easy‐to‐run method for routine analysis of eumelanin and pheomelanin in pigmented tissues

A procedure for analysis of melanin‐pigmented tissues based on alkaline hydrogen peroxide degradation coupled with high‐performance liquid chromatography (HPLC) ultraviolet determination of pyrrole‐2,3,5‐tricarboxylic acid (PTCA) for eumelanin and 6‐(2‐amino‐2‐carboxyethyl)‐2‐carboxy‐4‐hydroxybenzothiazole (BTCA) and 1,3‐thiazole‐2,4,5‐tricarboxylic acid for pheomelanin was recently developed. Despite advantages related to the degradation conditions and sample handling, a decrease of the reproducibility and resolution was observed after several chromatographic runs. We report herein an improved chromatographic methodology for simultaneous determination of PTCA and BTCA as representative markers of eumelanin and pheomelanin, respectively, based on the use of an octadecylsilane column with polar end‐capping with 1% formic acid (pH 2.8)/methanol as the eluant. The method requires conventional HPLC equipments and gives very good peak shapes and resolution, without need of ion pair reagents or high salt concentrations in the mobile phase. The intra‐assay precision of the analytical runs was satisfactory with CV values ≤4.0% (n = 5) for the two markers which did not exceed 8% after 50 consecutive injections on the column over 1 week. The peak area ratios at 254 and 280 nm (A₂₈₀/A₂₅₄: PTCA = 1.1, BTCA = 0.6) proved a valuable parameter for reliable identification of the structural markers even in the most complex degradation mixtures. The method can be applied to various eumelanin and pheomelanin pigmented tissues, including mammalian hair, skin and irides, and is amenable to be employed in population screening studies.     

A double EPSPS gene mutation endowing glyphosate resistance shows a remarkably high resistance cost

A novel glyphosate resistance double point mutation (T102I/P106S, TIPS) in the 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) gene has been recently identified for the first time only in the weed species Eleusine indica. Quantification of plant resistance cost associated with the TIPS and the often reported glyphosate resistance single P106S mutation was performed. A significant resistance cost (50% in seed number currency) associated with the homozygous TIPS but not the homozygous P106S EPSPS variant was identified in E. indica plants. The resistance cost associated with the TIPS mutation escalated to 85% in plants under resource competition with rice crops. The resistance cost was not detected in nonhomozygous TIPS plants denoting the recessive nature of the cost associated with the TIPS allele. An excess of 11‐fold more shikimate and sixfold more quinate in the shikimate pathway was detected in TIPS plants in the absence of glyphosate treatment compared to wild type, whereas no changes in these compounds were observed in P106S plants when compared to wild type. TIPS plants show altered metabolite levels in several other metabolic pathways that may account for the expression of the observed resistance cost.     

Genetic analysis of pathway regulation for enhancing branched‐chain amino acid biosynthesis in plants

The branched‐chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that play critical roles in animal growth and development. Animals cannot synthesize these amino acids and must obtain them from their diet. Plants are the ultimate source of these essential nutrients, and they synthesize BCAAs through a conserved pathway that is inhibited by its end products. This feedback inhibition has prevented scientists from engineering plants that accumulate high levels of BCAAs by simply over‐expressing the respective biosynthetic genes. To identify components critical for this feedback regulation, we performed a genetic screen for Arabidopsis mutants that exhibit enhanced resistance to BCAAs. Multiple dominant allelic mutations in the VALINE‐TOLERANT 1 (VAT1) gene were identified that conferred plant resistance to valine inhibition. Map‐based cloning revealed that VAT1 encodes a regulatory subunit of acetohydroxy acid synthase (AHAS), the first committed enzyme in the BCAA biosynthesis pathway. The VAT1 gene is highly expressed in young, rapidly growing tissues. When reconstituted with the catalytic subunit in vitro, the vat1 mutant‐containing AHAS holoenzyme exhibits increased resistance to valine. Importantly, transgenic plants expressing the mutated vat1 gene exhibit valine tolerance and accumulate higher levels of BCAAs. Our studies not only uncovered regulatory characteristics of plant AHAS, but also identified a method to enhance BCAA accumulation in crop plants that will significantly enhance the nutritional value of food and feed.     

A special reactor design for investigations of mixing time effects in a scaled‐down industrial L‐lysine fed‐batch fermentation process

A specially designed model reactor based on a 42‐L laboratory fermentor was equipped with six stirrers (Rushton turbines) and five cylindrical disks. In this model reactor, the mixing time, Θ₉₀, turned out to be 13 times longer compared with the 42‐L standard laboratory fermentor fitted with two Rushton turbines and four wall‐fixed longitudinal baffles. To prove the suitability of the model reactor for scaledown studies of mixing‐time‐dependent processes, parallel exponential fed‐batch cultivations were carried out with the leucine‐auxotrophic strain, Corynebacterium glutamicum DSM 5715, serving as a microbial test system. L‐ Leucine, the process‐limiting substrate, was fed onto the liquid surface of both reactors. Cultivations were conducted using the same inoculum material and equal oxygen supply. The model reactor showed reduced sugar consumption (−14%), reduced ammonium consumption (−19%), and reduced biomass formation (−7%), which resulted in a decrease in L ‐lysine formation (−12%). These findings were reflected in less specific enzyme activity, which was determined for citrate synthase (CS), phosphoenolpyruvate carboxylase (PEP‐C), and aspartate kinase (AK). The reduced specific activity of CS correlated with lower CO₂ evolution (−36%) during cultivation. The model reactor represents a valuable tool to simulate the conditions of poor mixing and inhomogeneous substrate distribution in bioreactors of industrial scale. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 599–606, 1999.     

High‐performance liquid chromatography evaluation of the enantiomeric purity of amino acids by means of automated precolumn derivatization with ortho‐phthalaldehyde and chiral thiols

The use of ortho‐phthalaldehyde (OPA) for the derivatization of amino acids (AA) is well known. It enables the separation of the derivatives on common reversed phase columns and improves the sensitivity with fluorescence detection. With the use of a chiral thiol an indirect enantioseparation of chiral amines and AAs is feasible. The major drawback of the OPA‐derivatization is the poor stability of the products. Here, a method with an in‐needle derivatization procedure is optimized to facilitate a quantitative conversion of the AA with OPA and the chiral thiols N‐acetyl‐L‐cysteine or N‐isobutyryl‐L‐cysteine, followed by a subsequent analysis, eluding the stability issue. Both enantiomers of a single AA were separated as OPA‐derivatives with a pentafluorophenyl column and a gradient program consisting of 50 mM sodium acetate buffer pH = 5.0 and acetonitrile. Fluorescence detection is commonly used to achieve sufficient sensitivity. In this study, the enantiomeric impurity of an AA can be detected indirectly with common UV spectrophotometric detection with a limit of quantitation of 0.04%. Seventeen different L‐AAs were tested and the amount of D‐AA for each individual AA was calculated by means of area normalization, which ranged from not detectable up to 4.29%. The recovery of the minor enantiomer of L‐ and D‐AA was demonstrated for three AAs at a 0.04% level and ranged between 92.3 and 113.3%, with the relative standard deviation between 1.7 and 8.2%.     

A convenient microwave‐enhanced solid‐phase synthesis of short chain N‐methyl‐rich peptides

Structural modification of the peptide backbone via N‐methylation is a powerful tool to modulate the pharmacokinetic profile and biological activity of peptides. Here we describe a rapid and highly efficient microwave(MW)‐assisted Fmoc/tBu solid‐phase method to prepare short chain N‐methyl‐rich peptides, using Rink amide p‐methylbenzhydrylamine (MBHA) resin as solid‐phase support. This method produces peptides in high yield and purity, and reduces the time required for Fmoc‐N‐methyl amino acid coupling. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.