Aqueous Two-phase Partitioning Research Articles

Assaying the Effect of Peptide Treatment on H+-Pumping Activity in Plasma Membranes from Arabidopsis Seedlings.

Extracellular acidification or alkalization is a common response to many plant-signaling peptides and microbial elicitors. This may be a result of peptide-mediated regulation of plasma membrane-localized ion transporters, such as the plasma membrane H+-ATPase. Early responses to some signaling peptides can therefore be analyzed by assaying H+-pumping across the plasma membrane.We describe a set-up suited for the purification of plasma membranes by aqueous two-phase partitioning from a small sample of Arabidopsis seedlings. Seedlings are grown in a liquid culture, suited for the analysis of in vivo peptide treatment. Additionally, we describe how to measure the H+-pumping activity of the plasma membrane H+-ATPase using the fluorescent probe ACMA.

Characteristics and Application of Lipase from Asian Seabass Liver Fractionated Using Aqueous Two-phase Partition Technique for Defatting Fish Skin before Collagen Extraction

Lipase, from crude extract of fish viscera including liver, is highly contaminated with other hydrolytic enzymes such as proteases that would cause severe degradation of native collagen during defatting. Asian seabass liver lipase was therefore subjected to fractionation with the aqueous two-phase system (ATPS) to remove proteases from lipase. ATPS was carried out using various salts and polyethylene glycol (PEG) having various molecular weights at different concentrations. The concentration of 20% ammonium sulfate (w/v) and 50% PEG-6000 (w/w) could reduce protease by 85%. Lipase-rich fraction showed a specific activity of 68 U/mg protein, purification fold of 9, selectivity of 167, and yield of 48%. When pulse electric field (PEF) was treated Asian seabass fish skin was defatted, and the acid-soluble collagen was extracted and characterized. The ATPS fractionated lipase could remove more than 92% of lipid contents. Polyunsaturated and monounsaturated fatty acids were mainly eliminated. The extracted collagen from defatted skin showed typical type Ⅰ collagen with negligible degradation. FTIR spectra substantiated the presence of amide groups in resulting skin collagen. Thus, the fractionation of Asian seabass liver crude extract with ATPS could significantly remove the contaminated proteases. The obtained fraction could be used to defat Asian seabass skin without drastic damage to the extracted native collagen.

Tyrosinase from the peels of Musa cavendish: purification, characterization, immobilization and application in phenol biodegradation

Tyrosinase is an enzyme implicated in fruit ripening and the browning of plant parts exposed to mechanical injury. Tyrosinase has numerous reported biotechnological importance. This enzyme was extracted from the waste peels of Musa cavendish, purified via aqueous two-phase partitioning system (ATPS) and QAE-Sephadex ion-exchange chromatography. The purified tyrosinase was biochemically characterized. Tyrosinase was thereafter immobilized, characterized, and applied in the oxidation of phenols in model wastewaters. After purification, the yield and fold obtained were 23% and 8.0 respectively. The molecular weight of the enzyme was estimated to be 43.0 kDa. The purified enzyme had an optimum pH and temperature of 6.5 and 60 °C respectively. The (M−1s−1) obtained for substrates such as L-DOPA, catechol and pyrogallol were respectively. The purified enzyme was stable in organic solvents: acetone > ethanol > methanol > n-butanol. Quercetin, ascorbic acid, l-cysteine and arginine were potent inhibitors. The immobilized tyrosinase was more resistant to thermoinactivation than the free enzyme as evidenced by its kinetic ( and thermodynamic ( characteristics. The immobilized tyrosinase removed 70% of 650-µM phenol after 2 h compared to 50% removal by the free tyrosinase. After six repeated cycles of phenol biodegradation using the immobilized enzyme, about 40% of phenol was removed. The fresh peels of M. cavendish served as a green-based source of relatively thermostable immobilized tyrosinase deployable in large-scale biodegradation of phenol-containing wastewater.

Biochemical properties of purified polyphenol oxidase from bitter leaf (Vernoniaamygdalina)

Polyphenol oxidase which is responsible for oxidative conversion of phenolic compounds to polymers, has continued to attract the attention of scientists. Here, we report the extraction, purification and biochemical properties of polyphenol oxidase (PPO) from bitter leaf (Vernonia amygdalina). The enzyme was purified and concentrated using a non-conventional approach, aqueous two-phase partitioning (ATPS) and the biochemical properties of the purified enzyme were investigated. Substrate specificity studies revealed that the enzyme predominantly exhibits diphenolase activity. The order of substrate preference was catechol > L-DOPA > caffeic acid > L-tyrosine > resorcinol>2-naphthol > phenol. The optimum pH and temperature obtained for the enzyme using catechol as substrate were 5.5 and 50 °C respectively. The estimated Michaelis constant (Km) and maximum velocity (Vmax) for the purified vaPPO using catechol as substrate were 183 ± 5.0 mM and 2000 ± 15 units/mg protein respectively. The catalytic efficiency (Vmax/Km) of the purified vaPPO was 10.9 ± 0.03 min/mg. Na+, K+ and Ba2+ remarkably activated the enzyme and the level of activation was proportional to the concentration. The vaPPO presented stability in the presence of up to 50 mM of the different metal ions tested. In contrast, Cu2+ and NH4+ inhibited the enzyme even 10 mM concentrations. The enzyme was stable in chloroform retaining up to 60% relative activity at 50% (v/v) concentration. There was an increase in the activity (143%) of the enzyme at 30% (v/v) chloroform., revealing that vaPPO could catalyze the substrate more efficiently in 30% (v/v) chloroform. Total loss of enzyme activity was observed at 20% (v/v) concentrations of acetone, ethanol and methanol.In conclusion, the properties of the vaPPO such as its catalysis in the presence of organic solvents, metals and high temperature would be of interest in many biotechnological applications.

Novel peroxidase from bitter leaf (Vernonia amygdalina): Purification, biochemical characterization and biotechnological applications

Bitter leaf could serve as a cost-effective and alternative source of plant peroxidase for several applications. In this study, the bioavailability, biochemical properties and potentials of bitter leaf peroxidase in some biotechnological applications are described. The enzyme was first purified using a fast and cost-effective approach. The combination of aqueous two-phase partitioning and gel filtration chromatography on Bio Gel P-100 was very efficient resulting to a percentage yield and final purification fold of 76% and 7.1 respectively. Native and subunit molecular weights of the purified peroxidase were 41.2 ± 2.1 kDa and 39.5 ± 1.0 kDa, respectively, revealing the monomeric nature of bitter leaf POD. The optimum pH was 6.0 while that of temperature was 30 °C. The estimated kinetic constants Km were 2.5 ± 0.1 mM and 5.1 ± 0.2 mM for o-dianisidine and hydrogen peroxide respectively while maximum velocity (Vmax) was 3010.6 ± 21 units/mg protein. The enzyme was activated in the presence of organic solvents (at up to 30% chloroform). It also retained stability (≥60%) at up to 50% concentration of acetone, methanol and ether. Chlorides of Ca2+, Na+ and Cu2+ activated the enzyme whereas Al3+ and Fe3+ had inhibitory effects. Bitter leaf POD was covalently bonded with casein to form higher molecular adduct, establishing its potentials as a reporter enzyme. In conclusion, bitter leaf peroxidase possesses remarkable biochemical properties that could be of interest in several industrial and biotechnological applications.

Effect of Temperature on the Phase Equilibria of Poly(vinylpyrrolidone) 10000 + Trilithium Citrate + Water Aqueous Two-Phase System and Partitioning of Curcumin at T = 298.15, 308.15, and 318.15 K

Effect of Temperature on the Phase Equilibria of Poly(vinylpyrrolidone) 10000 + Trilithium Citrate + Water Aqueous Two-Phase System and Partitioning of Curcumin at <i>T</i> = 298.15, 308.15, and 318.15 K

Potentials of purified tyrosinase from yam (Dioscorea spp) as a biocatalyst in the synthesis of cross-linked protein networks

We report the usefulness of yam tyrosinase as a catalyst in the synthesis of cross-linked protein networks for biopolymers. The enzyme was purified using aqueous two-phase partitioning (ATPs) and peptide mapping on SDS-PAGE was carried out to ascertain degree of similarities of tyrosinase from the yam species. The mapping revealed distinct peptide bands of 3, 4, 4 and 2 for tyrosinase from D. praehensilis, D. alata, D. rotundata and C. esculenta respectively purified using conventional method. In contrast, continuous broad band was noticed for the ATPS-purified enzymes due to bound polyethylene glycol (PEG). Tyrosinase from D. praehensilis with overall better properties was used in the synthesis of cross-linked protein networks. The enzyme catalyzed conversion of soluble proteins from whey, moringa leaves, pumpkin leaves and cow blood into fibrous (cross-linked) protein networks for improved properties and functionalities. The purified tyrosinase from D. praehensilis was also covalently bonded to bovine serum albumin (BSA) forming tyrosinase-BSA adduct with molecular weight of 118 ± 2.0 kDa, revealing its potential as a reporter enzyme by reporting BSA. The overall result further reinforces yam tyrosinase as an enzyme of interest in various biotechnological applications.

Aqueous two-phase partitioning and characterization of xylanase produced by Streptomyces geysiriensis from low cost lignocellulosic substrates

Microbial production of xylanase is gaining the commercial importance, due to its wide range of applications from paper and pulp to food and feed industries. Streptomyces geysiriensis was used for the production of extracellular xylanase from lignocellulosic substrates such as rice bran and saw dust, under solid-state fermentation. The influence of pH, temperature and incubation period for the maximum production of xylanase was investigated with 1:2 (w/v) of substrate to moisture ratio at 100rpm shaking conditions. The maximum production was recorded after 5 days of fermentation with pH 8.0at 40°C. The scale-up was done based on the results of optimized parameters using 3L Applikon autoclavable bioreactor with maximum yield of 186 U/ml after 4 days of fermentation. Extracellular xylanase was separated by partitioning in aqueous two-phase system consisting of 20% polyethylene glycol 6000 and 12% K2HPO4 with maximum yield of 93.97%. The investigation of the effect of pH and temperature and its incubation time showed that xylanase was retained its activity in a pH range of 6.5-8.5, with thermal stability from 20°C to 60°C up to 180min. The presence of metal ions was found to inhibit the activity of xylanase especially Cu2+ and Zn2+. Xylanase was stable both at 4°C and room temperature (35°C) for 30 and 9 days respectively. The kinetic parameters Km (0.48mg/ml) and Vmax (8.33 U/mg) were determined using birchwood xylan as substrate.

Physicochemical properties of free and immobilized tyrosinase from different species of yam (Dioscorea spp)

A shortened method of purification and immobilization of tyrosinase from different species of yam (Dioscorea spp) on insoluble supports is described. The enzyme was purified by aqueous two-phase partitioning (ATPS) followed by gel filtration chromatography. The purified enzyme was immobilized on Ca-alginate, polyacrylamide gel or as cross-linked enzyme aggregate (CLEA) to obtain a yield of between 51–64%, 33–46% and 52–65% respectively for all the yam species. The optimum pH obtained for tyrosinase immobilized on polyacrylamide gel and CLEA was equivalent to that of free enzyme (pH 6.5). In contrast, Ca-alginate entrapped tyrosinase exhibited a shift of optimum pH to 7.0. Entrapped Tyrosinase in polyacrylamide gel and Ca-alginate also retained the same optimum temperature as the free enzyme (50 °C). While the optimum temperature of CLEA shifted to 60 °C. When subjected to four repeated use cycles, tyrosinase entrapped in polyacrylamide gel, Ca-alginate and CLEA still retained close to 40, 35 and 45 % of their initial activities respectively after the fourth cycle. The overall result further suggests yam tyrosinase as a promising enzyme for biocatalysis and biotechnological applications.

Characterization of low temperature-induced plasma membrane lipidome remodeling combined with gene expression analysis reveals mechanisms that regulate membrane lipid desaturation in Carica papaya

Carica papaya is a cold-sensitive horticultural crop, the mechanisms underlying its cold sensitivity remains unclear which limit its genetic improvements in cold tolerance. In this study, aqueous dextran-polyethylene glycol two-phase partition systems were applied to purify plasma membrane from papaya leaves grown under 28 °C and 10 °C, the plasma membrane total lipids were analyzed by lipidomic approach; meanwhile, the transcription levels of putative fatty acid desaturases were quantified by RT-qPCR. We found that cerebrosides are dominate plasma membrane lipid species, followed by phospholipids and free sterols. Cold treatment increased the mono-unsaturated and di-unsaturated phospholipids, and decreased di-saturated phospholipids, the proportion of total phospholipids was not affected. Cold stress enhanced the transcription of CpFAD4, CpFAD6 and CpFAB2−2. Pathway analysis suggested that the cold induction of CpFAB2−2 transcription and CpFAD2 enzyme activities play important roles for plasma membrane lipidome remodeling under cold stress. Our findings uncovered potential targets to improve papaya cold tolerance.

A new tool for quantification of membrane protein partitioning between different cell membranes

The aqueous two-phase partition system (ATPS) is a method widely used to separate and purify plant and animal membranes carrying bound proteins. However, a common problem of this separation is a mutual contamination of obtained phases. Such contamination adversely affects the accuracy of values of the protein of interest partition between particular membranes when determined by direct measurement. In order to overcome this problem, we have developed a fairly simple mathematical algorithm and found formulas designed to quantify correctly the distribution of the protein of interest between two different membranes. This new tool makes it possible to determine the bias-adjusted ratio of protein distribution between the membranes, regardless of the efficiency of membrane separation in a two-phase system. By means of this algorithm, not only current, but also a number of previously published ATPS-based experiments were (re)analyzed and quantified. The quantitative results of this large-scale analysis of the subcellular localization of various membrane proteins from Arabidopsis, potato, melon, and corn including cytokinin and ethylene receptors, ABCG14 cytokinin transporters, LRR receptor-like protein kinases, and BAK1 co-receptors are presented and discussed here.

Evidences for Preferential Localization of Cytokinin Receptors of Potato in the Endoplasmic Reticulum

Cytokinins, classical plant hormones, are recognized by the cell through receptors—hybrid sensor histidine kinases, multidomain transmembrane proteins. The character of the hormonal effect on the cell and the mode of the hormonal signal transduction to the primary targets depends on the subcellular localization of the receptors. Recently, we cloned and investigated a family of cytokinin receptors from a new model plant, potato Solanum tuberosum L. Two typical representatives of this family, StHK2a and StHK4b, were selected to study their subcellular localization. In the current study, total membranes isolated from tobacco leaves, in which the selected recombinant potato receptors fused to GFP-reporter were transiently expressed, were fractionated by aqueous two-phase partitioning. Two membrane fractions were obtained, one tentatively enriched with endoplasmic reticulum (ER) membranes and the other, with the plasma membrane (PM). The content of StHK2a and StHK4b in each fraction was determined by the radioligand method. Both receptors were detected in the two fractions, with a slight predominance of StHK4b in the ER-enriched fraction. Given that the content of ER membranes in the cell is markedly higher than the content of the PM, the bulk of the receptors should be attributed to the ER. Specific cytokinin-binding sites were detected in the analogous membrane fractions isolated from non-transformed potato roots. Although the specific content of the cytokinin-binding sites was 1.8-fold higher in the PM, here also, the main part of these sites, i.e., cellular receptors, still resided in the ER. In order to find out whether the receptors in the ER are functional, a recently proposed pH criterion was applied. According to this criterion, potato cytokinin receptors, like their orthologs from other species, seem to be tuned for functioning in the ER but not in the PM. The results of molecular modeling of the dimerization interface of the receptors at different pH values also evidenced in favor of the ER. Thus, the main site of subcellular localization of the functionally active potato cytokinin receptors is the ER, although a certain amount of the receptors resides within the PM, too. The question of the functional role of PM-located receptors remains open.

Production of adeno-associated virus vectors for in vitro and in vivo applications

Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Of note, we achieved titers of 1010–1011 viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. Our study provides an improved protocol for a more economical and efficient purified AAV preparation.

Optimization of aqueous two-phase partitioning for purification of recombinant Eupenicillium terrenum fructosyl peptide oxidase

Abstract Fructosyl peptide oxidase (FPOX), a flavoenzyme belonging to the class of oxidoreductases, has been used as a diagnostic enzyme for HbA1c measurement test. Design of experiments methodology was applied to evaluate the partition behavior of recombinant Eupenicillium terrenum FPOX in aqueous two-phase systems (ATPS). Different ATPS parameters including polyethylene glycol (PEG) molecular weight, PEG concentration, salt type, salt concentration, pH, system buffer type, system buffer concentration, temperature, neutral salt type, neutral salt concentration, and crude enzyme loading were investigated using a Plackett–Burman design (PBD). Taking into consideration an increase in partition coefficient (KE), PEG concentration, pH, system buffer concentration, and NaCl concentration, which played significant roles in enzyme partition, were chosen for further optimization by Box–Behnken design (BBD) in response surface methodology (RSM). The optimized condition led to maximum recovery (106.8%), yield (95.1%) and purification factor (42.4) values which were predicted to be achieved in ATPS formed by 14.0% (w/w) PEG-4000, 15.0% (w/w) Na2CO3, 5.0% (w/w) NaCl and 55.0 mM potassium phosphate buffer with pH 8.0. The partitioned recombinant FPOX was obtained as a single band into the upper PEG-rich phase and its specific activity was calculated to be 201.23 ± 2.1 U/mg. In summary, our data showed that the ATPS optimization using design of experiments can be an appropriate method for recovery of recombinant FPOX.

Optimization of aqueous two-phase partitioning of Aureobasidium pullulans α-amylase via response surface methodology and investigation of its thermodynamic and kinetic properties

Industrial enzymes such as α-amylase must be thermostable and also easily purified/concentrated. Hence, aqueous two-phase partitioning systems (ATPS) was exploited for the partitioning of α-amylase from Aureobasidium pullulans due to its numerous advantages over conventional purification strategy. A. pullulans α-amylase was partially purified using ATPS via response surface methodology (RSM). The potentials of the ATPS-purified enzyme for possible industrial application such as resistance to thermal inactivation was investigated in comparison with the crude enzyme. PEG-6000 was the polymer of choice for ATPS as it resulted in higher purification factor (PF), %yield (Y), and partition coefficient (PC). At optimum levels (% w/v) of 20, 12 and 7.5 for PEG-6000, sodium citrate and sodium chloride respectively, maximum PF, Y and PC of 4.2, 88%, and 9.9 respectively were obtained. The response model validation and reliability were established based on the closeness between the experimented and predicted values. The kinetic and thermodynamic parameters such as Q10, t1/2, kd, D − value, Ed, ΔHd∗,ΔGd∗andΔSd∗ of the ATPS-purified α-amylase indicated that it was thermostable at 50 to 60 °C compared to the crude α-amylase. A thermodynamically stable and ATPS-purified α-amylase from A. pullulans has properties easily applicable for most industrial processes.

Plasma Membrane Proteomic Profile Discovers Macrophage-capping Protein Related to Latent HIV-1.

Due to the persistence of latent HIV-infected cellular reservoirs, HIV virus can not be eradicated completely. To identify proteins related to HIV latency, we performed a subcellular proteomic study in HIV latent cell lines. An established HIV-1 latent cell model (J-Lat Tat-GFP Clone A7 cells, A7 cells) and its parental cell line (Jurkat cells) were used. The plasma membrane (PM) fraction from cultured cells was enriched through aqueous two-phase partition. PM proteins were extracted and then separated using two-dimensional electrophoresis (2DE). Differentially expressed proteins were identified by mass spectrometry, and verified by western blotting. Thirteen non-redundant proteins were identified to be differentially expressed in the A7 PM fraction compared to those in the Jurkat PM. Eight had a PM location through Gene Ontology (GO) analysis. A differential protein network of CAPG-ACTR3-CD3D was detected to have interactions with HIV Vpr, Tat, gp160, etc. through STRING software analysis. One of the differential proteins (Macrophage-capping protein (CAPG)) was verified by western blotting to be down- regulated in two cell lines and HIV resting CD4+ T cells negatively selected from patients. We identified 13 proteins in A7 compared to Jurkat cells. CAPG may be a potential biomarker related to HIV latency.

Use of Aqueous Two-Phase and Three-Phase Partitioning Systems for Purification of Lipase Obtained in Solid-State Fermentation by Rhizopus arrhizus

Background: Purification of enzymes by conventional methods such as precipitation and chromatographic techniques is a costly and time-consuming procedure and may lead to low yields of enzyme activity. Alternative liquid-liquid extraction methods such as Aqueous Two-Phase Systems (ATPS) and Three Phase Partitioning (TPP) are characterized by the high enzyme yields and purification degree. Objective: The objective of this study was the application of partitioning systems ATPS and TPP for purification of lipase produced in solid-state fermentation by Rhizopus arrhizus. Methods: ATPS and TPP were used for purification of lipase, obtained by solid state cultivation of Rhizopus arrhizus. Results: Lipase was isolated with PEG4000/potassium sodium tartrate ATPS and the effect of the system composition, including PEG 4000 and potassium sodium tartrate concentrations on lipase partitioning was studied. When using 30% PEG4000/21% potassium sodium tartrate, lipase was distributed in the top phase, and the highest recovery yield of 217% and purification fold of 6.1 were achieved. It was found that at PEG4000 concentration of or higher than 15%, the enzyme was present in the top polymer-rich phase with a partitioning yield of over 90%. Upon application of TPP for lipase isolation, the effect of t-butanol concentration, ammonium sulfate concentration and pH on enzyme partitioning was investigated. The highest lipase recovery yield of 71% and 19.1-fold purification were achieved in the interfacial phase in the presence of 30% ammonium sulfate saturation with 1.0:0.5 crude extract/t-butanol ratio at pH 7 in a single step. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymographic analysis showed significant purification of lipase by TPP and the presence of two multiple forms of the enzyme. Conclusion: ATPS (PEG4000/ Potassium sodium tartrate) and TPP (1.0:0.5 crude extract/t-butanol ratio, 30% ammonium sulfate saturation, pH 7) proved to be rapid methods for the isolation and purification of lipase and they can be used in downstream processing for industrial preparation of the enzyme.

Partial characterization of Bacillus pumilus catalase partitioned in poly(ethylene glycol)/sodium sulfate aqueous two-phase systems

Aqueous two-phase partitioning system (ATPS) was used to extract and purify catalase from Bacillus pumilus. The system parameters for effective purification of catalase were optimized. The best catalase recovery (123%) with a 4.6-fold purification was obtained in the bottom phase of ATPS including the mixture of 15% (w/w) PEG4000, 10% (w/w) Na2SO4 and 3% (w/w) NaCl at pH 5.0. The purified enzyme was characterized regarding its activity and stability. The highest enzyme activity was observed at pH 7.0 and 37 °C on hydrogen peroxide. The enzyme was quite stable at temperatures between 30 and 55 °C and a pH range of 7.0–9.0. The Km and Vmax values were determined from Lineweaver–Burk plot as 11 mM and 1667 µmole ml−1 min−1, respectively. Overall, it can be said that ATPS is a rapid, reasonable, straightforward and cost-effective process for catalase purification in comparison to the chromatographic methods.

13 - Use of adeno-associated viruses to inhibit Glutaredoxin-1 in mouse skeletal muscle

Glutaredoxin-1 (Glrx) is a cytosolic enzyme that reverses glutathione (GSH) adducts of protein thiols generated through oxidative post-translational modifications. GSH-adducts regulate cellular signaling and transcription factors. We previously found that after femoral artery ligation Glrx knockout mice improved blood flow recovery in relation to activation of hypoxia-inducible factor (HIF)-1α via GSH adducts, indicating Glrx as an anti-angiogenic enzyme. Objective We hypothesized that local Glrx gene inhibition might improve poor blood flow recovery after ischemia in aging or diabetes. To inhibit Glrx gene expression in vitro and in vivo, we developed an improved protocol to produce adeno-associated viruses (AAVs) with a bicistronic expression system coding for Glrx shRNA (shGlrx) and mVenus, a yellow fluorescent protein. Methods Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation, aqueous two-phase partitioning, and an iodixanol gradient, which resulted in AAVs with adequate titer and purity for in vivo use. We tested the AAVs on C2C12 skeletal muscle cells and mouse gastrocnemius muscles. Results Quiescent confluent C2C12 cells transduced with a shGlrx-containing AAV exhibited a decrease of ~75% in the Glrx mRNA and protein levels after 4 days. Intramuscular injection (4.4 x 1012 viral genome/50 μl) of AAV-shGlrx transduced mVenus expression in skeletal muscle efficiently, but non-injected muscle, heart, or liver showed no mVenus expression. However, 4 weeks after intramuscular injection, Glrx protein levels remained unchanged compared to control-AAV injected or non-injected muscle, and inflammatory markers were increased. Six weeks after virus injection, Glrx expression in the muscle decreased 80% at the mRNA and 50% at the protein level. Conclusion Intramuscular injection of AAV-shGlrx resulted in muscle-specific attenuation of Glrx expression after at least 6 weeks. AAV intramuscular injection caused local inflammation in the early stage which may counteract the effects of shGlrx. (R01HL133013, R03AG051857, R01DK103750)

Factorial and Economic Evaluation of an Aqueous Two-Phase Partitioning Pilot Plant for Invertase Recovery From Spent Brewery Yeast.

Aqueous two-phase systems (ATPS) have been reported as an attractive biocompatible extraction system for recovery and purification of biological products. In this work, the implementation, characterization, and optimization (operational and economic) of invertase extraction from spent brewery yeast in a semi-automatized pilot plant using ATPS is reported. Gentian violet was used as tracer for the selection of phase composition through phase entrainment minimization. Yeast suspension was chosen as a complex cell matrix model for the recovery of the industrial relevant enzyme invertase. Flow rates of phases did not have an effect, given that a bottom continuous phase is given, while load of sample and number of agitators improved the recovery of the enzyme. The best combination of factors reached a recovery of 129.35 ± 2.76% and a purification factor of 4.98 ± 1.10 in the bottom phase of a PEG-Phosphate system, also resulting in the removal of inhibitor molecules increasing invertase activity as reported by several other authors. Then, an economic analysis was performed to study the production cost of invertase analyzing only the significant parameters for production. Results indicate that the parameters being analyzed only affect the production cost per enzymatic unit, while variations in the cost per batch are not significant. Moreover, only the sample load is significant, which, combined with operational optimization results, gives the same optimal result for operation, maximizing recovery yield (15% of sample load and 1 static mixer). Overall res ults of these case studies show continuous pilot-scale ATPS as a viable and reproducible extraction/purification system for high added-value biological compounds.