Bioengineering of Bacteria To Assemble Custom-Made Polyester Affinity Resins

Proof of concept for the in vivo bacterial production of a polyester resin displaying various customizable affinity protein binding domains is provided. This was achieved by engineering various protein binding domains into a bacterial polyester-synthesizing enzyme. Affinity binding domains based on various structural folds and derived from molecular libraries were used to demonstrate the potential of this technique. Designed ankyrin repeat proteins (DARPins), engineered OB-fold domains (OBodies), and V_HH domains from camelid antibodies (nanobodies) were employed. The respective resins were produced in a single bacterial fermentation step, and a simple purification protocol was developed. Purified resins were suitable for most lab-scale affinity chromatography purposes. All of the affinity domains tested produced polyester beads with specific affinity for the target protein. The binding capacity of these affinity resins ranged from 90 to 600 nmol of protein per wet gram of polyester affinity resin, enabling purification of a recombinant protein target from a complex bacterial cell lysate up to a purity level of 96% in one step. The polyester resin was efficiently produced by conventional lab-scale shake flask fermentation, resulting in bacteria accumulating up to 55% of their cellular dry weight as polyester. A further proof of concept demonstrating the practicality of this technique was obtained through the intracellular coproduction of a specific affinity resin and its target. This enables in vivo binding and purification of the coproduced “target protein.” Overall, this study provides evidence for the use of molecular engineering of polyester synthases toward the microbial production of specific bioseparation resins implementing previously selected binding domains.     

Interaction Between Oxytocin and Opioidergic System on Food Intake Regulation in Neonatal Layer Type Chicken

The aim of the current study was to determine possible interaction of central oxytocin and opioidergic system on food intake regulation in neonatal layer-type chicken. In experiment 1, FD₃ chicken ICV injected with control solution, oxytocin (10 µg), β-FNA (µ receptor antagonist, 5 µg) and oxytocin (10 µg)?+?β-FNA were injected. Experiments 2–6 were similar to experiments 1, except chicken injected with nor-BNI (κ receptor antagonist, 5 µg), NTI (δ receptor antagonist, 5 µg), DAMGO (µ receptor agonist, 62.25 pmol), U-50488H (κ receptor agonist, 10 nmol), DPDPE (δ receptor agonist, 20 pmol) instead of β-FNA. In experiment 7, control solution, DAMGO (125 pmol), d(CH2)5Tyr(Me)-[Orn8]-vasotocin (oxytocin antagonist, 5 µg) and DAMGO?+?d(CH2)5Tyr(Me)-[Orn8]-vasotocin were ICV injected to FD₃ chicken. Experiments 8 and 9 were similar to experiments 7, except chicken injected with U-50488H (30 nmol) and DPDPE (40 pmol) instead of DAMGO. Then, cumulative food intake was recorded at 30, 60 and 120 min after injection. According to the results, ICV injection of the oxytocin (10 µg) significantly decreased food intake compared to control group (P?<?0.05). Co-injection of the oxytocin?+?β-FNA and oxytocin?+?U-50488H significantly decreased hypophagic effect of the oxytocin (P?<?0.05). While, co-injection of the oxytocin?+?nor-BNI or oxytocin?+?DAMGO significantly amplified hypophagic effect of the oxytocin in chicken (P?<?0.05). In addition, ICV injection of DAMGO (125 pmol) significantly decreased cumulative food intake compared to control group (P?<?0.05). However, co-addministration of the DAMGO?+?(CH2)5Tyr(Me)-[Orn8]-vasotocin significantly decreased hypophagic effect of the DAMGO (P?<?0.05) in chicken. These results suggested there are interconnection between oxytocin and opioidergic system on central food intake regulation, which mediates via µ and κ opioidergic receptors in neonatal layer-type chicken.

     

Interactions of 5-(1-Pyrenyl)-10,15,20-tris(4-methylpyridinium)porphine with Double-Helix and Triple-Helix Nucleic Acids

Abstract

5-(1 -Pyrenyl)-10,15,20-tris(4-methylpyridinium)porphine (H₂PTMPP) having a porphyrin ring and a pyrenyl substituent was synthesized. The compound H₂PTMPP bound to poly(dA)?poly(dT) double helix and poly(dA)?2poly(dT) triple helix in different styles. The results of H₂PTMPP binding to oligonucleotides, dA₁₄?dT₁₄ and dA₁₄?2dT₁₄, was also shown.

     

Drying techniques for the visualisation of agarose‐based chromatography media by scanning electron microscopy

The drying of chromatography resins prior to scanning electron microscopy is critical to image resolution and hence understanding of the bead structure at sub‐micron level. Achieving suitable drying conditions is especially important with agarose‐based chromatography resins, as over‐drying may cause artefact formation, bead damage and alterations to ultrastructural properties; and under‐drying does not provide sufficient resolution for visualization under SEM. This paper compares and contrasts the effects of two drying techniques, critical point drying and freeze drying, on the morphology of two agarose based resins (MabSelect?/d _w ≈85 µm and Capto? Adhere/d _w ≈75 µm) and provides a complete method for both. The results show that critical point drying provides better drying and subsequently clearer ultrastructural visualization of both resins under SEM. Under this protocol both the polymer fibers (thickness ≈20 nm) and the pore sizes (diameter ≈100 nm) are clearly visible. Freeze drying is shown to cause bead damage to both resins, but to different extents. MabSelect resin encounters extensive bead fragmentation, whilst Capto Adhere resin undergoes partial bead disintegration, corresponding with the greater extent of agarose crosslinking and strength of this resin. While freeze drying appears to be the less favorable option for ultrastructural visualization of chromatography resin, it should be noted that the extent of fracturing caused by the freeze drying process may provide some insight into the mechanical properties of agarose‐based chromatography media.     

Physicochemical and chromatographic evaluation of benzhydrylamine-resin as an anion exchanger solid support

Benzhydrylamine-resin (BHAR), a copolymer of styrene-divinylbenzene containing phenylmethylamine groups, used as solid support for peptide synthesis, was examined regarding physicochemical and anion exchanger chromatographic properties. The greater the ionic strength of the medium the poorer the solvation of beads. This effect is less pronounced the higher the amino group content of BHAR. The BHAR chromatographic behavior was compared with commercial cationic resins in columns of constant cation binding capacity. Three negatively charged heparan sulfate disaccharides were successfully purified in a 2.4 mmol/g BHAR that showed as good or better anion exchange performance than classical tertiary or quaternary amino group-containing resins. The BHAR chromatographic resin exclusion limit was estimated to be 30 kDa based on purification experiments of heparins of different molecular weight.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: γ-AMINOBUTYRIC ACID BINDING IN THE RAT CEREBRAL CORTEX

—The binding of [¹⁴C]GABA to nerve-ending membranes isolated from rat cerebral cortex follows a hyperbolic curve saturating at 0·4pmol/μg protein. This binding is about 60% inhibited by chloropromazine, and about 40%, inhibited by bicuculline. A hydrophobic protein fraction binding [¹⁴C]GABA was separated from the total. lipid extract of nerve-ending membranes. The binding follows a hyperbolic curve that saturates at 10·5 pmol of [¹⁴C]GABA/μg of protein, with an apparent K_d= 30 μm . The binding is competitively inhibited by bicuculline with a K_i= 273 μm . These results are compared with those previously obtained on a GABA binding protein from crustacean muscle.     

Evaluation of a novel methacrylate‐based protein a resin for the purification of immunoglobulins and Fc‐fusion proteins

Protein A affinity chromatography is a central part of most commercial monoclonal antibody and Fc‐fusion protein purification processes. In the last couple years an increasing number of new Protein A technologies have emerged. One of these new Protein A technologies consists of a novel, alkaline‐tolerant, Protein A ligand coupled to a macroporous polymethacrylate base matrix that has been optimized for immunoglobulin (Ig) G capture. The resin is interesting from a technology perspective because the particle size and pore distribution of the base beads are reported to have been optimized for high IgG binding and fast mass transfer, while the Protein A ligand has been engineered for enhanced alkaline tolerance. This resin was subjected to a number of technical studies including evaluating dynamic and static binding capacities, alkaline stability, Protein A leachate propensity, impurity clearance, and pressure–flow behavior. The results demonstrated similar static binding capacities as those achieved with industry standard agarose Protein A resins, but marginally lower dynamic binding capacities. Removal of impurities from the process stream, particularly host cell proteins, was molecule dependent, but in most instances matched the performance of the agarose resins. This resin was stable in 0.1 M NaOH for at least 100 h with little loss in binding capacity, with Protein A ligand leakage levels comparable to values for the agarose resins. Pressure–flow experiments in lab‐scale chromatography columns demonstrated minimal resin compression at typical manufacturing flow rates. Prediction of resin compression in manufacturing scale columns did not suggest any pressure limitations upon scale up. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1125–1136, 2014     

Solubilization and partial purification of the GABA receptor from mouse brain and a binding assay for the solubilized receptor.

Abstract— The GABA receptor from mouse brain was solubilized with lysolecithin. A 56-fold overall purification and activation were achieved by discontinuous sucrose gradient centrifugation and solubilization. Activation of binding by both procedures was observed. The solubilized receptor has the following binding constants: K_D1= 3.5 _nM, K_D2= 52 _nM, B_{max 1}= 2.8 pmol/mg protein and B_{max 2}= 14 pmol/mg protein for muscimol; K_D1= 12 nM, K_D2= 470 nM, B_{max 1}= 1.4 pmol/mg protein and B_{max 2}= 17 pmol/mg protein for GABA. Specific GABA binding was inhibited by imidazoleacetic acid and bicuculline with IC₅₀ values of 250_nM and 1 _μM respectively. A rapid and sensitive filtration binding assay for the solubilized receptor has been developed. Lysolecithin was also found suitable for the solubilization of acetylcholine receptor from T. californica electroplaques.     

Regioselective hydroxylation of cholecalciferol,cholesterol and other sterol derivatives by steroid C25 dehydrogenase

Steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S is a molybdenum oxidoreductase belonging to the so-called ethylbenzene dehydrogenase (EBDH)-like subclass of DMSO reductases capable of the regioselective hydroxylation of cholesterol or cholecalciferol to 25-hydroxy products. Both products are important biologically active molecules: 25-hydroxycholesterol is responsible for a complex regulatory function in the immunological system, while 25-hydroxycholecalciferol (calcifediol) is the activated form of vitamin D₃ used in the treatment of rickets and other calcium disorders. Studies revealed that the optimal enzymatic synthesis proceeds in fed-batch reactors under anaerobic conditions, with 6–9 % (w/v) 2-hydroxypropyl-β-cyclodextrin as a solubilizer and 1.25–5 % (v/v) 2-methoxyethanol as an organic co-solvent, both adjusted to the substrate type, and 8–15 mM K₃[Fe(CN)₆] as an electron acceptor. Such thorough optimization of the reaction conditions resulted in high product concentrations: 0.8 g/L for 25-hydroxycholesterol, 1.4 g/L for calcifediol and 2.2 g/L for 25-hydroxy-3-ketosterols. Although the purification protocol yields approximately 2.3 mg of pure S25DH from 30 g of wet cell mass (specific activity of 14 nmol min⁻¹ mg⁻¹), the non-purified crude extract or enzyme preparation can be readily used for the regioselective hydroxylation of both cholesterol and cholecalciferol. On the other hand, pure S25DH can be efficiently immobilized either on powder or a monolithic silica support functionalized with an organic linker providing NH₂ groups for enzyme covalent binding. Although such immobilization reduced the enzyme initial activity more than twofold it extended S25DH catalytic lifetime under working conditions at least 3.5 times.

     

CO2 reduction potentials by utilizing waste plastics in steel works

Background, aim, and scope  Feedstock recycling has received attention as an effective method to recycle waste plastics. However, estimating the reduction potential by life cycle assessment using coke oven and blast furnace in steel works has been a challenging task due to the complex structure of energy flow in steel works. Municipal waste plastics consist of several plastic resins. Previous studies have generally disregarded the composition of waste plastics, which varies significantly depending on the geographical area. If the reduction potentials by using each plastic resin in steel works can be quantified, the potential of municipal waste plastics (mixtures of plastic resins) can be estimated by summing up the potential of each resin multiplied by the composition of each resin in municipal waste plastics. Therefore, the goal of this study is to investigate the reduction potentials of CO₂ emissions by using individual plastic resins (polyethylene (PE), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET)) and those for municipal waste plastics in the coke oven and blast furnace. Materials and methods  A model was developed to clarify the energy flow in steel works. In order to estimate the changes in energy and material balance in coke ovens when waste plastics are charged, the equations to calculate the coke product yield, gas product yield, and oil product yields of each plastic resin were derived from previous studies. The Rist model was adopted to quantify the changes in the inputs and outputs when plastics were fed into a blast furnace. Then, a matrix calculation method was used to calculate the change in energy balance before and after plastics are fed into a coke oven. Results  It was confirmed that product yields of municipal waste plastics (mixtures of plastic resins) could be estimated by summing up the product yield of each plastic resin multiplied by the composition of each resin in municipal waste plastics. In both cases of coke oven and blast furnace feedstock recycling, the reduction potential of CO₂ emissions varies significantly depending on the plastic resins. For example, in the case of coke oven chemical feedstock recycling, the reduction potential of PS and PP is larger than that of PE. On the other hand, in the case of blast furnace feedstock recycling, PE has the largest CO₂ emissions reduction potential, whereas the CO₂ emission reduction potential of PP is smaller than those of PE and PS. In both cases, PET has negative CO₂ emission reduction potentials, i.e., there is an increase of CO₂ emissions. In addition, the reduction potentials of CO₂ emissions are slightly different in each city. Discussions  The differences in the reduction potentials of CO₂ emissions by coke oven chemical feedstock recycling of each plastic resin is attributable to the differences in calorific values and coke product yields of each plastic resin. On the other hand, the difference in the CO₂ emission reduction potential for each plastic resin in blast furnace feedstock recycling is attributable to the difference in calorific values and the carbon and hydrogen content of each plastic resin, which leads to a difference in the coke substitution effect by each plastic resin. In both cases, the difference in those of municipal waste plastics is mostly attributable to the amount of impurities (e.g., ash, water) in the municipal waste plastics. Conclusions  It was found that the reduction potential of CO₂ emissions by coke oven and blast furnace feedstock recycling of municipal waste plastics (mixtures of plastic resins) could be estimated by summing up the potential of each resin multiplied by the composition of each resin in municipal waste plastics. It was also clarified that feedstock recycling of waste plastic in steel works is effective for avoiding the increase in CO₂ emissions by incinerating waste plastics, such as those from household mixtures of different resins. Recommendations and perspectives  With the results obtained in this study, reduction potentials of CO₂ emissions can be calculated for any waste plastics because differences in composition are taken into account.     

Affinity resins containing enzymatically resistant mRNA cap analogs--a new tool for the analysis of cap-binding proteins

Cap-binding proteins have been routinely isolated using m⁷GTP-Sepharose; however, this resin is inefficient for proteins such as DcpS (scavenger decapping enzyme), which interacts not only with the 7-methylguanosine, but also with the second cap base. In addition, DcpS purification may be hindered by the reduced resin capacity due to the ability of DcpS to hydrolyze m⁷GTP. Here, we report the synthesis of new affinity resins, m⁷GpCH₂pp- and m⁷GpCH₂ppA-Sepharoses, with attached cap analogs resistant to hydrolysis by DcpS. Biochemical tests showed that these matrices, as well as a hydrolyzable m⁷GpppA-Sepharose, bind recombinant mouse eIF4E^(28-217) specifically and at high capacity. In addition, purification of cap-binding proteins from yeast extracts confirmed the presence of all expected cap-binding proteins, including DcpS in the case of m⁷GpCH₂pp- and m⁷GpCH₂ppA-Sepharoses. In contrast, binding studies in vitro demonstrated that recombinant human DcpS efficiently bound only m⁷GpCH₂ppA-Sepharose. Our data prove the applicability of these novel resins, especially m⁷GpCH₂ppA-Sepharose, in biochemical studies such as the isolation and identification of cap-binding proteins from different organisms.     

Synthesis and characterization of iron(llI) chelating analogues of siderophores on organic solid supports

A series of iron(III)-selective chelating resins have been modeled after the structural features of the naturally occurring siderophore compounds with hydroxamate, catecholate and salicylate iron binding groups. Amberlite IRC-50 was derivatized via an acid chloride intermediate to produce poly(hydroxamic acid) (IRC-50 PHA/N-H and IRC-50 PHA/N- CH₃), poly(catecholate) (IRC-50 PEDA 2,3-DHBAD and IRC-50 PEDA 3,4-DHBA) and poly(salicylate) (IRC-50 PEDA 2-HBAD) chelating resins with enhanced iron(III) binding capacity. The poly(hydroxamic acid) IRC-50 PHA/N-CH₃ was produced in 57% yield, the highest conversion yet reported for such derivatizations. This is the first report of a catechol or salicylate derivatization of Amberlite IRC-50. The highest overall iron(III) binding capacities yet reported for poly(hydroxamic acid) modifications of commercially available polymer supports were obtained for IRC-50 PHA/N-H (1.75 mmol Fe/g dry resin) and IRC-50 PHA/N-CH₃ (1.52 mmol Fe/g dry resin). IRC-50 PHA/N-H was also found to be an effective iron chelator when tested at ambient environmental conditions. Selectivity for iron- (III) was also determined by measuring Fe³⁺ binding capacity in the presence of Ca²⁺. UVVis spectroscopy with photoacoustic detection was used to assign the coordination environment of iron(III) in these chelating resins as bis-(FeL₂) and tris-(FeL₃) chelates. As an alternative synthetic approach, a poly(amidoxime) (PAO) chelating resin was synthesized by polymerization of appropriate monomers. Comparison between the two synthetic approaches showed that PAO bound a greater amount of iron(III) at flow-through column conditions, while IRC-50 PHA/N-H exhibited a greater iron(III) binding capacity at batch equilibration conditions.     

Preparation of purified vaccine from local isolate of foot and mouth disease virus and its immune response in bovine calves

Foot and Mouth Disease (FMD) is globally pandemic which badly affect the economics of livestock based countries like Pakistan. There are different types of Foot and Mouth Disease Virus (FMDV) among these types O is most prevalent in Pakistan. Recently Pakistan is producing approximately fifteen million doses of non-purified FMD vaccine against the demand of 160 million doses annually. More over the Pakistan is still striving for the development and optimization of concentration as well as purification of FMDV. The present project was designed to develop the technology for the purification of FMDV indigenously. The locally isolated and adapted FMDV type O virus was propagated on adherent culture of BHK-21cells to get final volume of virus one liter. This virus suspension was concentrated by peggylation as well as ultra-filtration method. The purification and quantification of concentrated virus was done by size exclusion chromatography. The results showed that peggylation is better method of concentration up to 603.75 µg/ml with 82.80 % recovery rate than ultra-filtration with 43.90 % followed by chromatography for purification. The PD₅₀ was calculated in bovines at 24, 12, 6, 3 and 1.5 µg of FMDV Ag/dose and it revealed that antigen load of 1.98 µg is the dose, where the 50 % of inoculated animals showed the protective antibody level based upon percent inhibition through antibody detecting ELISA. According to the British pharmacopeia, the vaccine should contain 3PD₅₀ which found equivalent to our findings about 6 µg/dose. The group of animal injected with 6/dose (3.23PD50) showed protective titer up to 20th week post priming.     

Enhanced production of periplasmic interferon alpha-2b by Escherichia coli using ion-exchange resin for in situ removal of acetate in the culture

The possibility of using in situ addition of anion-exchange resin for the removal of acetate in the culture aimed at improving growth of E. coli and expression of periplasmic human interferon-α2b (PrIFN-α2b) was studied in shake flask culture and stirred tank bioreactor. Different types of anion-exchange resin were evaluated and the concentration of anion-exchange resin was optimized using response surface methodology. The addition of anion-exchange resins reduced acetate accumulation in the culture, which in turn, improved growth of E. coli and enhanced PrIFN-α2b expression. The presence of anion-exchange resins did not influence the physiology of the cells. The weak base anion-exchange resins, which have higher affinity towards acetate, yielded higher PrIFN-α2b expression as compared to strong anion-exchange resins. High concentrations of anion-exchange resin showed inhibitory effect towards growth of E. coli as well as the expression of PrIFN-α2b. The maximum yield of PrIFN-α2b in shake flask culture (501.8 μg/L) and stirred tank bioreactor (578.8 μg/L) was obtained at ion exchange resin (WA 30) concentration of 12.2 g/L. The production of PrIFN-α2b in stirred tank bioreactor with the addition of ion exchange resin was about 1.8-fold higher than that obtained in fermentation without ion exchange resin (318.4 μg/L).     

Plasmid DNA purification using a multimodal chromatography resin

Multimodal chromatography is widely used for isolation of proteins because it often results in improved selectivity compared to conventional separation resins. The binding potential and chromatographic behavior of plasmid DNA have here been examined on a Capto Adhere resin. Capto Adhere is a recent multimodal chromatography material allowing molecular recognition between the ligand and target molecule, which is based on combined ionic and aromatic interactions. Capto Adhere proved to offer a very strong binding of nucleic acids. This property could be used to isolate plasmid DNA from a crude Escherichia coli extract. Using a stepwise NaCl gradient, pure plasmid DNA could be obtained without protein and endotoxin contaminations. The RNA fraction bound most strongly to the resin and could be eluted only at very high salt concentrations (2.0 M NaCl). The chromatographic separation behavior was very robust between pH values 6 and 9, and the dynamic binding capacity was estimated to 60 µg/ml resin. Copyright © 2014 John Wiley & Sons, Ltd.     

25-Hydroxycholecalciferol-binding protein: Partial purification from rat duodenal mucosa cells

The 25-hydroxycholecalciferol-binding protein has been partially purified (purification factor: 37) from rat duodenal cytosol, using chromatographic procedures on gel and ionic exchange resin. This partially purified protein bound 25-hydroxycholecalciferol with high affinity (K_D = 5.7 × 10^?9M) and low binding capacity (23 × 10^?12 mole/mg of protein. Using a rabbit antiserum obtained against such partially purified protein, we demonstrated that 25-hydroxycholecalciferol cytosolic binder and 25-hydroxycholecalciferol plasmatic binding share common antigenic sites.     

Multi-targeted dihydrazones as potent biotherapeutics

Hydrazone compounds were considered as a useful moiety in drug design development. Therefore, these studies were aimed at the synthesis of new dihydrazones and were screened for their in vitro H⁺/K⁺-ATPase and anti-inflammatory activities. The results revealed that compounds 9 (22 ± 0.62 µg/mL), 10 (26 ± 0.91 µg/mL), 15 (24 ± 0.44 µg/mL), 16 (28 ± 0.63 µg/mL), 17 (12 ± 0.38 µg/mL), 18 (14 ± 0.47 µg/mL), 19 (26 ± 0.54 µg/mL), 20 (16 ± 0.41 µg/mL), 25 (06 ± 0.68 µg/mL) and 26 (08 ± 0.43 µg/mL) showed excellent H⁺/K⁺-ATPase activity and their IC₅₀ value were lower than the standard drug Omerazole (48 ± 0.12 µg/mL). Compounds 5 (28 ± 0.65 µg/mL), 6 (24 ± 0.61 µg/mL), 7 (28 ± 0.64 µg/mL), 8 (26 ± 0.45 µg/mL), 11 (30 ± 0.74 µg/mL), 12 (28 ± 0.40 µg/mL), 13 (32 ± 0.24 µg/mL), 14 (30 ± 0.55 µg/mL) and 21 (08 ± 0.47 µg/mL), 22 (12 ± 0.47 µg/mL), 23 (10 ± 0.51 µg/mL) and 24 (14 ± 0.84 µg/mL) showed better anti-inflammatory activity compared to standard indomethacin (44 ± 0.15 µg/mL). The structure activity relationship (SAR) showed that, electron donating groups (OH, OCH₃) favored the H⁺/K⁺-ATPase and antioxidants activity, whereas, electron withdrawing groups (F, Cl, Br and NO₂) favored the anti-inflammatory activity. Furthermore, molecular docking study was performed to investigate the binding interactions of the most active analogs with the active site of H⁺/K⁺-ATPase enzyme. Compounds 25 (G-score = −9.063) and 26 (G-score = −8.977) showed the highest docking G-scores for H⁺/K⁺-ATPase inhibition activity.     

Development of a micelle-fractional precipitation hybrid process for the pre-purification of paclitaxel from plant cell cultures

A micelle-fractional precipitation hybrid process was developed for the effective pre-purification of the anticancer agent paclitaxel extracted from plant cell cultures. First, it was found that the efficiency of such a developed process could be remarkably enhanced by removing waxy substances originating from plant cells using the adsorbent sylopute. Paclitaxel yield was improved and the fractional precipitation time was shortened by increasing the surface area per working volume (S/V) of the reacting solution through the addition of a cation exchange resin (Amberlite IR120 or Amberlite 200), an anion exchange resin (Amberlite IRA400 or Amberlite IRA96), or glass beads. Most of the paclitaxel (>98%) could be obtained after about 12 h of fractional precipitation using Amberlite 200. Purity increased with increasing fractional precipitation time up to 9 h to about 85%, after which it showed little change. On the other hand, no paclitaxel precipitate was formed using either of the nonionic exchange resins because paclitaxel, which is hydrophobic, was strongly adsorbed on the hydrophobic resin surface. Since high-purity paclitaxel can be obtained in high yield and the precipitation time can be reduced by combining micelle formation with fractional precipitation, this hybrid method is expected to significantly enhance the final purification process.     

Development of simple and rapid elution methods for proteins from various affinity beads for their direct MALDI-TOF downstream application

Commercially available desalting techniques, necessary for downstream MALDI-TOF analysis of proteins, are often costly or time consuming for large-scale analysis. Here, we present techniques to elute proteins from various affinity resins, free from salt and ready for MALDI mass spectrometry. We showed that 0.1% TFA in 50% acetonitrile or 40% ethanol can be used as salt-free eluents for His-tagged proteins from variety of polyhistidine-affinity resins, while washing of resin beads twice with double-distilled water prior to the elution effectively desalted and recovered wide-range-molecular size proteins than commercially available desalting devices. Modified desalting and elution techniques were also applied for Flag- and Myc-tag affinity resins. The technique was further applied in co-precipitation assay, where the maximum recovery of wide-range molecular size proteins is crucial. Further, results showed that simple washing of the beads with double distilled water followed by elution with acetonitrile effectively desalted and recovered 150 kDa factor H protein of the sheep and its binding partner ~30 kDa BbCRASP-1 in co-precipitation assay. In summary, simple modifications in the desalting and elution strategy save time, labor and cost of the protein preparation for MALDI mass spectrometry; and large-scale protein purifications or co-precipitations can be performed with ease.     

Interaction of glucocorticoid receptor from rat liver with protamine and arginine

The nontransformed glucocorticoid receptor (GR) from rat liver was found to bind to protamine-Sepharose and could be recovered by a salt gradient without a change in molecular configuration. The nontransformed GR also bound to arginine-Sepharose, but the transformed GR did not bind to either resin. Ligand-free GR interacted with both resins and was eluted without loss of its steroid binding ability. The bindings of GR to protamine- and arginine-Sepharose were saturable. The apparent dissociation constants of GR on protamine-Sepharose varied from 0.34 nM (−molybdate) to 0.68 nM (+10 mM molybdate) and those on arginine-Sepharose were 1.99 nM (− molybdate) and 0.65 nM (+ 10 mM molybdate), respectively. The maximum binding capacity was achieved by arginine-Sepharose in the absence of molybdate. Higher salt concentrations (0.5 M NaCl) were required to elute GR from protamine-Sepharose than from arginine-Sepharose (approx 0.03 M NaCl). However, the effectiveness of several salts for the elution of GR was consistent in both resins as follows; MgCl₂ = CaCl₂ = Na₂WO₄ > (NH₄)₂SO₄ = Na₂MoO₄ > arginine-HCl > lysine-HCl > KCl = NaCl. These results suggest that GR interacts with arginine residues in protamine. Chromatography using these resins resulted in 7–10-fold purification of occupied and unoccupied nontransformed GRs.