Application of electrochemically produced aluminum hydroxide gel for prepurification of recombinant synthetic green fluorescent protein produced in tobacco leaves

The use of recombinant proteins has increased greatly in recent years, as also have increased the number of techniques and materials used for their production and purification. Among the different types of bioreactors being studied, there is a general consensus among scientists that production in green plant tissues such as leaves is more feasible. However, the presence of chlorophyll and phenolic compounds in plant extracts, which can precipitate and denature the proteins besides damaging separation membranes and gels, makes this technology impracticable on a commercial scale. In the present work, the adsorption to electrochemically produced aluminum hydroxide gel was applied as a prepurification step for recombinant synthetic green fluorescent protein (sGFP), also referred to as enhanced green fluorescent protein, produced in Nicotiana benthamiana leaves. Removal efficiencies of 99.7% of chlorophyll, 88.5% of phenolic compounds, and 38.5% of native proteins from the N. benthamiana extracts were achieved without removing sGFP from the extracts. As electrochemical preparation of aluminum hydroxide gel is a cost‐effective technique, its use can substantially contribute to the development of future production platforms for recombinant proteins produced in green plant tissues of pharmaceutical and industrial interest. © 2011 American Institute of Chemical Engineers Biotechnol. Prog.,, 2011     

Modeling of protein and phenolic compound removal from aqueous solutions by electrocoagulation

Electrocoagulation is a technique basically applied in water and wastewater treatment, but which has a number of potential applications in polymer, protein, drug, and vaccine delivery. In this work, we correlate the current applied between the electrodes to the removal of phenolic compounds or protein from aqueous solutions, but the principle can also be applied to other biological compounds such as plant pigments and sugars. Simple and time‐dependent models were developed based on the complex formation between these biological substances and the aluminium hydroxide gel phase. The models developed represent a good agreement with experimental data (R² as high as 0.992). Besides construction of the models, the effect of pH on the efficiency of removal of proteins and phenolic compounds was evaluated. It was found that this parameter has significant effect on the efficiency of the electrocoagulation and the maximal removal efficiency for bovine serum albumin and phenolic compound catechin was observed at pH 8.0. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The use of Aspergillus niger for the bioconversion of olive mill waste-waters

Summary Olive mill waste-water was used for protein production in small-scale experiments, using non-sterilized medium without pH control. A 14 g/1 concentration of proteins, 61% chemical oxygen demand removal and a 58% reduction in total phenolic compounds were obtained using an Aspergillus niger strain. The removal of phenolic compounds resulted in a change in the colour of the waste-water from black to brown.Offprint requests to: J. L. Garcia     

Aqueous extraction of recombinant human proinsulin from transgenic maize endosperm

Different plant species have been used as systems to produce recombinant proteins. Maize is a crop considered to have a large potential to produce high levels of recombinant proteins and is the host for the recombinant proteins from plants currently available on the market. In the development of a plant system to produce a recombinant proteins it is important to consider the costs related to downstream processing. Also, the steps necessary to achieve the protein purity required will be highly influenced by the quality of the extract obtained. In this study, we analyzed aqueous extracts from the endosperm of transgenic maize expressing recombinant human proinsulin (rhProinsulin). A study of the effects of the variables pH and ionic strength on the extraction efficiency was carried out using experimental design and response surface methodology. Besides the concentration of the recombinant protein, the characteristics of the extracts were evaluated in terms of concentration of native components (proteins, carbohydrates, and phenolic compounds) and extract filterability. The highest rhProinsulin concentration (97.33 ng/mL) was found with a 200 mM NaCl pH 10.0 extraction solution. Under this experimental condition the concentrations of total soluble proteins, carbohydrates, and phenolics were 2.01 mg/mL, 2.21 mg/mL, and 0.11 mmol/L, respectively.     

Slaughterhouse wastewater treatment by combined chemical coagulation and electrocoagulation process

Slaughterhouse wastewater contains various and high amounts of organic matter (e.g., proteins, blood, fat and lard). In order to produce an effluent suitable for stream discharge, chemical coagulation and electrocoagulation techniques have been particularly explored at the laboratory pilot scale for organic compounds removal from slaughterhouse effluent. The purpose of this work was to investigate the feasibility of treating cattle-slaughterhouse wastewater by combined chemical coagulation and electrocoagulation process to achieve the required standards. The influence of the operating variables such as coagulant dose, electrical potential and reaction time on the removal efficiencies of major pollutants was determined. The rate of removal of pollutants linearly increased with increasing doses of PACl and applied voltage. COD and BOD(5) removal of more than 99% was obtained by adding 100 mg/L PACl and applied voltage 40 V. The experiments demonstrated the effectiveness of chemical and electrochemical techniques for the treatment of slaughterhouse wastewaters. Consequently, combined processes are inferred to be superior to electrocoagulation alone for the removal of both organic and inorganic compounds from cattle-slaughterhouse wastewater.     

Optimization of Protein Extraction from <Emphasis Type="Italic">Hypericum perforatum</Emphasis> Tissues and Immunoblotting Detection of Hyp-1 at Different Stages of Leaf Development

Sample preparation is crucial for obtaining high-quality proteins for the purpose of electrophoretic separation and further analysis from tissues that contain high levels of interfering compounds. Hypericum perforatum is a medicinal plant that contains high amounts of phenolic compounds, of which hypericins, hyperforins, and flavonoids contribute to the antidepressant activities of the plant. This study focuses on obtaining optimized amounts of high-quality proteins from H. perforatum, which are suitable for electrophoretic analyses. From the tested protein extraction solutions, sodium borate buffers at pH 9 and 10 gave the best protein yields from mature H. perforatum leaves. With these buffers, relatively high protein yields could also be obtained from roots, stems, and flower buds. The protein extracts of all organs were well resolved in SDS-PAGE after an efficient removal of non-protein contaminants with PVPP, phenol extraction, and methanolic ammonium acetate precipitation. The method was suitable for high-quality protein extraction also from other tested species of genus Hypericum. The applicability of the protocol for immunoblotting was demonstrated by detecting Hyp-1 in H. perforatum leaves at different stages of development. Hyp-1, which has been suggested to attend to the biosynthesis of hypericin, accumulated in high amounts in H. perforatum leaves at mature stage.     

Phenolics removal from transgenic Lemna minor extracts expressing mAb and impact on mAb production cost

Transgenic Lemna minor has been used successfully to produce several biotherapeutic proteins. For plant-produced mAbs specifically, the cost of protein A capture step is critical as the economic benefits of plant production systems could be erased if the downstream processing ends up being expensive. To avoid potential modification of mAb or fouling of expensive protein A resins, a rapid and efficient removal of phenolics from plant extracts is desirable. We identified major phenolics in Lemna extracts and evaluated their removal by adsorption to PVPP, XAD-4, IRA-402, and Q-Sepharose. Forms of apigenin, ferulic acid, and vitexin comprised ～ 75% of the total phenolics. Screening of the resins with pure ferulic acid and vitexin indicated that PVPP would not be efficient for phenolics removal. Analysis of the breakthrough fractions of phenolics adsorption to XAD-4, IRA-402, and Q-Sepharose showed differences in adsorption with pH and in the type of phenolics adsorbed. Superior dynamic binding capacities (DBC) were observed at pH 4.5 than at 7.5. To evaluate the cost impact of a phenolics removal step before protein A chromatography, a mAb purification process was simulated using SuperPro Designer 7.0. The economic analysis indicated that addition of a phenolics adsorption step would increase mAb production cost only 20% by using IRA-402 compared to 35% for XAD-4 resin. The cost of the adsorption step is offset by increasing the lifespan of protein A resin and a reduction of overall mAb production cost could be achieved by using a phenolics removal step.     

Improvement of fermentative decolorization of olive mill wastewater by Lactobacillus paracasei by cheese whey's addition

The effect of the cheese whey's (CW) addition on the fermentative decolorization of olive mill wastewater (OMW) by Lactobacillus paracasei, with and without pH adjustment by lime, was investigated. Mixtures OMW/CW at different proportions were fermented. The highest colour removal (47%) and total phenolic reduction (22.7%) of OMW were obtained after cofermentation of OMW/CW at proportions of 10/90, respectively. The decrease of pH after cofermentation of the two wastewaters, induced the precipitation of whey proteins with phenolic compounds and, so, improves decolorization. These removal yields reached 64% and 34%, respectively after precipitation by adjustment of pH at 7 with lime at the end of cofermentation. These improvements were correlated to a clarification of wastewaters by precipitation of whey proteins with phenolic compounds. An enhanced decolorization (up to 93%) and a total phenolic reduction (50%) of the mixture were obtained when cofermentation sequentially pH corrected by lime addition was investigated.     

缺铁逆境胁迫下水稻叶片蛋白的双向电泳及其蛋白质组图谱分析

水稻幼苗经缺铁胁迫诱导分别处理1、3、5天后,用酚法和TCA/丙酮法提取叶片中的可溶性蛋白进行双向电泳分析,从而研究在缺铁条件下叶片中蛋白表达的动态变化规律.结果显示1.不同pH IPG胶条分离蛋白的效果不同.用pH3-10的IPG胶条进行双向电泳,经考马斯亮蓝染色后,可在胶面上检测到大约450个蛋白点,其中约有89%的蛋白是酸性蛋白.如果用pH4-7的IPG胶条进行双向电泳,则可检测到大约600个蛋白点,其中有29个蛋白是上调表达,1个蛋白是下调表达,5个蛋白是诱导特异表达.2.不同方法提取的可溶性蛋白质量不同.TCA法简单易操作,似乎对于碱性蛋白的抽提效果更好,在2-DE图像上,减性端显示的蛋白点多;但此方法所得蛋白的再溶性差.酚法提取的蛋白再溶性好,所抽提的蛋白量较大,纯度较高.     

Carbonate extraction process for the metabolic, isozymic and proteomic profiling of rose-scented geranium (Pelargonium sp.), a hyper-acidic plant

Rose-scented geranium (Pelargonium sp.) is a valuable monoterpene-yielding plant. It has been well characterised phytochemically through the isolation of >270 secondary metabolites, however, there is hardly any biochemical or metabolic information concerning this plant. Initial attempts to investigate its metabolism failed to produce any enzyme activity in the tissue extracts prepared in routine extraction buffers owing to the intrinsic properties of the tissue matrix. It was recognised that cellular hyper-acidity (cell sap pH approximately 3.0) gave rise to very low protein levels in the extracts, thus prohibiting detection of activities of even primary metabolic enzymes that are usually abundantly present in plants. Tissue extraction in Tris solution without pH adjustment (as used for studies involving citrus and banana) led to little or no improvement. Therefore, a novel approach using sodium carbonate solution as an efficient extraction system for enzymes and proteins from the plant was studied. Functionality of the carbonate extraction has been demonstrated through its effectiveness, a several-fold superior performance, in yielding protein, monitoring primary metabolism and secondary metabolic enzymes, and isozymic and polypeptide profiling. The process may also be helpful in the reliable analysis of other acidic plant tissues.     

Escherichia coli HdeB is an acid stress chaperone

We cloned, expressed, and purified the hdeB gene product, which belongs to the hdeAB acid stress operon. We extracted HdeB from bacteria by the osmotic-shock procedure and purified it to homogeneity by ion-exchange chromatography and hydroxyapatite chromatography. Its identity was confirmed by mass spectrometry analysis. HdeB has a molecular mass of 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which matches its expected molecular mass. We purified the acid stress chaperone HdeA in parallel in order to compare the two chaperones. The hdeA and hdeB mutants both display reduced viability upon acid stress, and only the HdeA/HdeB expression plasmid can restore their viability to close to the wild-type level, suggesting that both proteins are required for optimal protection of the bacterial periplasm against acid stress. Periplasmic extracts from both mutants aggregate at acidic pH, suggesting that HdeA and HdeB are required for protein solubilization. At pH 2, the aggregation of periplasmic extracts is prevented by the addition of HdeA, as previously reported, but is only slightly reduced by HdeB. At pH 3, however, HdeB is more efficient than HdeA in preventing periplasmic-protein aggregation. The solubilization of several model substrate proteins at acidic pH supports the hypothesis that, in vitro, HdeA plays a major role in protein solubilization at pH 2 and that both proteins are involved in protein solubilization at pH 3. Like HdeA, HdeB exposes hydrophobic surfaces at acidic pH, in accordance with the appearance of its chaperone properties at acidic pH. HdeB, like HdeA, dissociates from dimers at neutral pH into monomers at acidic pHs, but its dissociation is complete at pH 3 whereas that of HdeA is complete at a more acidic pH. Thus, we can conclude that Escherichia coli possesses two acid stress chaperones that prevent periplasmic-protein aggregation at acidic pH.     

Polyphenol oxidase activity expression in Ralstonia solanacearum

Sequencing of the genome of Ralstonia solanacearum revealed several genes that putatively code for polyphenol oxidases (PPOs). To study the actual expression of these genes, we looked for and detected all kinds of PPO activities, including laccase, cresolase, and catechol oxidase activities, in cellular extracts of this microorganism. The conditions for the PPO assays were optimized for the phenolic substrate, pH, and sodium dodecyl sulfate concentration used. It was demonstrated that three different PPOs are expressed. The genes coding for the enzymes were unambiguously correlated with the enzymatic activities detected by generation of null mutations in the genes by using insertional mutagenesis with a suicide plasmid and estimating the changes in the levels of enzymatic activities compared to the levels in the wild-type strain. The protein encoded by the RSp1530 locus is a multicopper protein with laccase activity. Two other genes, RSc0337 and RSc1501, code for nonblue copper proteins exhibiting homology to tyrosinases. The product of RSc0337 has strong tyrosine hydroxylase activity, and it has been shown that this enzyme is involved in melanin synthesis by R. solanacearum. The product of the RSc1501 gene is an enzyme that shows a clear preference for oxidation of o-diphenols. Preliminary characterization of the mutants obtained indicated that PPOs expressed by R. solanacearum may participate in resistance to phenolic compounds since the mutants exhibited higher sensitivity to L-tyrosine than the wild-type strain. These results suggest a possible role in the pathogenic process to avoid plant resistance mechanisms involving the participation of phenolic compounds.     

Monoclonal antibodies to barley aleurain and homologs from other plants

Barley aleurain is contained within a specific type of vacuole characterized by acidic pH and the presence of other hydrolytic enzymes. The aleurain-containing vacuole is distinct from protein storage vacuoles, and anti-aleurain antibodies serve as markers for this organelle in barley cells. Aleurain is a unique type of cysteine protease, and other plant species have genes for homologs whose sequences are highly conserved, but little is known about these homologs at the protein level. Seven monoclonal antibodies to barley aleurain were isolated, which bind to and define aleurain homologues in Arabidopsis, Petunia , and tobacco cell extracts. Interestingly, in addition to 29–32 kDa aleurain homologs, Petunia extracts contain a protein of ∼50 kDa and tobacco extracts a protein of ∼40 kDa that are recognized by multiple different mono-clonal antibodies, indicating an unexpected diversity to the aleurain protein family. Among the group of antibodies are some that efficiently immunoprecipitate metabolically labeled aleurain from barley cell extracts, and some that efficiently label aleurain in immunofluorescence assays using root tip cells. These antibodies should be useful for plant cell biologists who study vacuole biogenesis and function and sorting of proteins to specific vacuolar compartments, in barley as well as other plant species.     

Chemical defense in birch: Inhibition of digestibility in ruminants by phenolic extracts

Summary The biological activity of phenolic extracts originating from winter twigs of birch (Betula pendula Roth.) was measured using the ruminant in vitro method and the nylon bag technique. Different extracts were prepared by extraction with organic solvents, removing phenols of corresponding solubility. The extract of birch twigs (diameter <1.5 mm) contained about 19% phenol equivalents, corresponding to 6% of twig dry matter (DM). Coarse birch twigs (diameter 1.5–5 mm) contained about 3% in the DM Phenolic extracts from the fine birch twigs were added to coarse birch twigs and common timothy (Phleum pratense L.) to mimic natural concentrations of fine birch twigs. Controls and the plant material with phenolic extract added were incubated for different times with rumen inocula taken from a sheep fed browse and a goat fed hay. Nylon bags containing phenolic treated hay were incubated in the rumen of the goat for 6 and 48 h. Phenolic extracts had a considerable negative effect on the organic matter (OM), protein and cell wall (neutral detergent fiber, NDF) digestibility in vitro. The nylon bag OM disappearance was also depressed by the extract. The effects were measurable after 6 h of digestion both in vitro and in sacco.The high inhibitory effect by the extracts on digestibility persisted even after removal of lipophilic fractions. This suggests that some or several water-soluble phenolic substances are responsible for the depression of digestibility. The depression of OM digestibility is linearly related to the concentration of phenols added. However, the inhibition of nylon bag digestibility plateaus at high phenol concentrations, suggesting that some fraction of the substances undergo complex formation with macromolecules of the plant.The results strongly indicate that water-soluble phenols of birch make up an important part of its chemical defense in winter by possessing antinutritional properties. Thus their potential importance in the nutrition of wild herbivores must not be ignored.     

Identification of target antigens of self‐reactive IgG in intravenous immunoglobulin preparations

Intravenous immunoglobulin (IVIg) contains a wide range of self‐reactive immunoglobulins (Ig) G. Acidic pH is known to increase the reactivity of purified IgG with self‐antigens. We describe here the target antigens of IgG autoantibodies in IVIg and analyze the influence of acidic pH on IgG reactivities. We used 2‐DE and immunoblotting with protein extracts of human umbilical vein endothelial cells (HUVEC) and HEp‐2 cells. Two IVIg preparations obtained by ethanol fractionation [one with an acidic pH step (acidic‐IVIg) and one with β‐propiolactone (propiolactone‐IVIg)] and a pool of sera from 12 healthy individuals were tested. Serum IgG of 3 healthy individuals and IgG purified from the same sera with elution at pH 2.8 were also tested individually. Finally, propiolactone‐IVIg was acidified at pH 2.8. IgG obtained with a step at low pH recognized many more target spots than IgG obtained without acidic pH. Our findings demonstrate that an acidic pH step artificially enlarges the repertoire of self‐reactive IgG. Thus, protein spots recognized by IgG in propiolactone‐IVIg represent the core set of self‐antigens targeted by IVIg. Overall, 96 proteins were identified by MS. Fourteen were recognized in both extracts including glycolysis proteins such as α‐enolase, RNA processing and cytoskeletal proteins such as lamin‐A/C.     

Fractionation of leaf proteins by differential centrifugation and gel filtration

The quantity and quality of fractionated leaf proteins from different plant species were investigated. Leaf extracts (pH range 7.0–8.3) were prepared on a laboratory scale from greenhouse cultivated plants. The proteins were fractionated by differential centrifugation followed by gel filtration. Amaranthus caudatus and Chenopodium quinoa seem favourable for production of non-green leaf protein concentrates, since the water-soluble protein comprised nearly 50% of the extracted protein. The chlorophyll-associated protein in fresh extracts from these species was, however, difficult to sediment. A practical separation of chlorophyll-associated protein from the chlorophyll-free proteins will require some kind of pre-aggregation of the chlorophyll-associated proteins. In extracts from Dactylis glomerata, Lolium perenne and Vicia sativa a considerable proportion of the extracted protein sedimented rapidly, but at least for the grasses some of the chlorophyll-associated protein remained in the supernatants even after centrifugation at 30 000 g for 180 min. Practical separation of all the chlorophyll-associated protein from the extract requires some kind of pre-aggregation of the chlorophyll-associated proteins even for these species. The situation was similar for extracts from Helianthus annuus, but a very high percentage of the total chlorophyll-associated proteins could be sedimented at low centrifugation speed. Brassica oleracea was the most suitable species for the removal of the chlorophyll-associated proteins by centrifugation alone. Moreover, the proportion of chlorophyll-free protein in the extracts was relatively high, more than 40%. The in vitro digestibility of membrane-bound protein fractions was high for species with co-aggregation of water-soluble protein and membrane-bound protein. Causes underlying the different distributions of the chlorophyll-associated and chlorophyll-free proteins and the reason for the different size of the chlorophyll-containing particles are discussed.     

Efficient solubilization buffers for two-dimensional gel electrophoresis of acidic and basic proteins extracted from wheat seeds

Plant tissues are made up of a broad range of proteins with a variety of properties. After extraction, solubilization of a diverse range of plant proteins for efficient proteomic analysis using two-dimensional electrophoresis is a challenging process. We tested the efficiency of 12 solubilization buffers in dissolving acidic and basic proteins extracted from mature seeds of wheat. The buffer containing two chaotropes (urea and thiourea), two detergents (3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate and N-decyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate), two reducing agents (dithiothreitol and tris (2-carboxyethyl) phosphine hydrochloride) and two types of carrier ampholytes (BioLyte pH 4-6 and pH 3-10) solubilized the most acidic proteins in the pH range between 4 and 7. The buffer made up of urea, thiourea, 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, DeStreak reagent (Amersham Biosciences, Uppsala, Sweden) and immobilized pH gradient buffer, pH 6-11 (Amersham Biosciences) solubilized the most basic proteins in the pH range between 6 and 11. These two buffers produced two-dimensional gels with high resolution, superior quality and maximum number of detectable protein (1425 acidic protein and 897 basic protein) spots.     

Evaluation of mutagenic activity in supply water at three sites in the state of Rio Grande do Sul, Brazil

In this study, the Salmonella/microsome assay, using the micro-suspension method, was utilized to evaluate water for public supply at three sites in Rio Grande do Sul. The first site selected was in an area under industrial influence and the others were in non-industrial reference areas. Based on 40 L samples of raw water and/or after conventional treatment, compounds were extracted with XAD4 resins using natural and acidic pH, and the extracts were analyzed in the TA98 and TA100 strains with and without S9. Raw water extracts in the industrial region induced 27.4 revertants/L (rev/L) for TA100+S9 up to 226.3 rev/L for TA100-S9, both for acidic pH extracts. After conventional treatment the responses varied from 20.6 rev/L (TA98-S9) for natural pH extracts to 755.5 rev/L (TA98-S9) for acidic pH extracts. For acidic extracts obtained from reference site samples, the response, with metabolic activation only, ranged from negative to minimal. Direct mutagenic responses in acidic extracts may be elevated in treated extracts, compared with raw water extracts, influenced by the presence of by-products of the chlorination process. However, the mutagenicity observed in the treated water extracts in the industrial area increased and reflected mainly the combination of directly and indirectly acting compounds in the source waters, that are heavily influenced by anthropogenic factors.     

加工因素对橄榄叶提取物抗氧化活性的影响

研究在橄榄叶加工过程中,加工因素对橄榄叶提取物中抗氧化活性的影响,这些因素包括:(1)干燥方式(微波炉干燥,烘箱干燥,低温冷冻干燥和熏蒸及烘箱结合干燥);(2)提取方式(超声波提取和搅拌式提取);(3)提取溶剂的酸度(pH 4、7、10).实验将总多酚含量和清除自由基活性作为衡量提取物抗氧化有效成分和活性的指标.研究结果表明,通过低温冷冻干燥、调节pH至4和采用超声波提取的方式,橄榄叶提取物中的抗氧化有效成分最多.本研究对橄榄叶等天然植物中抗氧化成分提取工艺的制定具有参考价值.     

A technique for the rapid removal of proteins from cell extracts containing polysaccharides

Summary The contaminating proteins in microbial cell extracts containing desired polysaccharides, can be rapidly precipitated by adjusting the pH of the solution. In the present research, 90% of the contaminating proteins in the alkali extract of Mucor rouxii were eliminated by lowering the pH, through addition of an organic acid. The acid for pH adjustment was chosen so that it formed a readymade buffer for use in subsequent chromatographic steps for recovery of the polysaccharide. The pH adjustment procedure thus offers a rapid and easily scaleable method for protein removal from cell extracts containing polysaccharides.