Hydrophobic Interaction Column Research Articles

Antiviral and Immunostimulating Effects of Lignin-Carbohydrate-Protein Complexes fromPimpinella anisum

Three antiviral and immunostimulating substances (LC1, LC2 and LC3) were isolated from a hot water extract of seeds of Pimpinella anisum by combination of anion-exchange, gel filtration and hydrophobic interaction column chromatographies. Chemical and spectroscopic analyses revealed them to be lignin-carbohydrate-protein complexes. These lignin-carbohydrate complexes (LCs) showed antiviral activities against herpes simplex virus types 1 and 2 (HSV-1 and -2), human cytomegalovirus (HCMV) and measles virus. LCs were also found to interfere with virus adsorption to the host cell surface and directly inactivate viruses. Furthermore, they enhanced nitric oxide (NO) production by inducing iNOS mRNA and protein expression in RAW 264.7 murine macrophage cells. The induced mRNA expression of cytokines including IL-1β and IL-10 was also apparent. These results suggest that the lignin-carbohydrate-protein complexes from P. anisum possessed potency as functional food ingredients against infectious diseases.

Calcium Hydroxide Inactivates Lipoteichoic Acid from Enterococcus faecalis through Deacylation of the Lipid Moiety

Introduction Lipoteichoic acid (LTA) is a major virulence factor of Enterococcus faecalis that is closely associated with refractory apical periodontitis. Recently, we have shown that calcium hydroxide, a commonly used intracanal medicament, abrogated the ability of LTA to stimulate the production of tumor necrosis factor α in a murine macrophage line, RAW 264.7. Because calcium hydroxide could potentially modify the glycolipid moiety of LTA, we examined if calcium hydroxide inactivates LTA through deacylation of the LTA. Methods LTA was prepared from E. faecalis by organic solvent extraction followed by chromatography with the hydrophobic-interaction column and the ion-exchange column. RAW 264.7 cells were stimulated with intact LTA or calcium hydroxide–treated LTA for 24 hours, and the productions of nitric oxide (NO) and chemokines interferon-gamma–induced protein (IP-10) and macrophage inflammatory protein-1α (MIP-1α) were determined. The glycolipid structure of LTA was analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry and thin layer chromatography (TLC). Results The production of NO, IP-10, and MIP-1α was augmented in LTA-stimulated cells, whereas no such effect was observed upon stimulation with calcium hydroxide–pretreated LTA. Mass spectrometry showed that intact glycolipids of LTA yielded distinct mass peaks at 930 to 1,070 mass over charge (m/z) units, corresponding to dihexosyl-diacylglycerol consisting of two acyl chains with chain lengths of C 16 to C 22 and with one or two unsaturated double bonds. However, those peaks were not observed in the mass spectra of the calcium hydroxide–treated LTA. Furthermore, free fatty acids released from the calcium hydroxide–treated LTA were detected using TLC. Conclusion We suggest that calcium hydroxide attenuates the inflammatory activity of E. faecalis LTA through deacylation of the LTA.

Identification of calpastatin, μ-calpain and m-calpain in Atlantic salmon ( Salmo salar L.) muscle

A soft fish muscle is generally considered as a poor quality trait among consumers and producers. This degradation and softening of post mortem muscle is thought to be partly caused by proteolytic enzymes such as the calpain system. Separation and identification of μ-calpain and m-calpain and their inhibitor – calpastatin, from Atlantic salmon ( Salmo salar) muscle were for the first time assessed in this study. A two-step chromatography approach was used, starting with a hydrophobic interaction column and followed by an anion exchange column. Calpastatin was successfully separated from calpain by hydrophobic interaction chromatography, and following the anion exchange chromatography, two forms of calpastatin (I and II) and two forms of calpain (micro (μ) and milli (m)) were revealed. The proteolytic activity of μ-calpain was detectable with column chromatography, but not consistently detected with casein zymography, and m-calpain was detected with both chromatography and casein zymogram. The proteolytic activity of m-calpain per g muscle was 15 times higher than that of μ-calpain. μ-Calpain had a temperature optimum of 15 °C and a maximum calcium requirement at 0.2 mM, while m-calpain had temperature optimum at 25 °C and a maximum calcium requirement of 0.6 mM. The two forms of calpastatin differed in inhibitory activity with calpastatin II having the highest activity. Both calpastatins tolerated heat treatment, as previously seen for mammals, and they kept their activity when stored at −80 °C, but not at −20 °C. The calpain to calpastatin ratio was 1:4.5 as observed for beef muscle. This study provides evidence that two calpain isoforms, likely to be μ- and m-calpain, in addition to two forms of calpastatin exist in Atlantic salmon muscle.

New method for preparation of hydrophobic interaction chromatographic stationary phases based on polymer‐grafted silica and their chromatographic properties

AbstractA new “grafting to” method for the preparation of polymer‐grafted silica hydrophobic interaction (HIC) packings was used in following steps. Firstly, 3‐mercaptopropyltrimethoxysilane was bonded on silica to obtain SH‐group modified silica. Secondly, glycidylmethacrylate (GMA) was coated on the surface of SH‐silica to obtain polymer‐grafted epoxide‐silica. Finally, the polymer‐grafted epoxide‐silica was dispersed in polyethylene glycol of dioxane solution to produce HIC stationary phase. The prepared HIC packings have advantages for biopolymer separation, high column efficiency and good resolution for proteins. The dynamic protein loading capacity of the synthesized packings was 36.0 mg/g. Six proteins were fast separated in less than 12.0 minutes using the synthesized HIC stationary phases. Then the stationary phase was evaluated in detail to determine the effects of pH of mobile phase, concentration of ammonium sulfate and the temperature on the retention of proteins. The thermodynamic parameters of these proteins are consistent with the entropically driven nature of hydrophobic interactions, The HIC column was used for purification of trypsin from a crude extract solution with only one step. The purity of the purified trypsin was more than 95%. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

Regulation of 1-aminocyclopropane-1-carboxylate oxidase gene expression during leaf ontogeny in white clover

The expression of two 1-aminocyclopropane-1-carboxylate (ACC) oxidase (E.C. 1.4.3)(ACO) genes, TR-ACO2 and TR-ACO3 from white clover has been examined in leaf tissue in response to physiological stimuli. In detached mature-green leaves, the expression of TR-ACO3 (a leaf-senescence-associated ACC oxidase) is induced over a 6 h time-course, while expression of TR-ACO2 (expressed constitutively in mature-green leaf tissue) decreased over the same time-course. An almost identical pattern of expression was observed with detached mature-green leaves treated with ethylene, while wounding also did not influence the timing of TR-ACO3 induction, significantly. No increase in overall ACO enzyme activity was detectable in crude extracts from wounded and detached leaf tissue, but after separation of these extracts using hydrophobic interaction column chromatography, two ACO isoforms were identified in non-wounded mature-green leaves (designated MGI and MGII), and the specific activity of one of these (MGII) increased in wounded tissues. In attached leaf tissue, TR-ACO2, but not TR-ACO3 expression was induced by treatment with ethylene; an induction that was not observed with prior treatment of plants with 1-MCP. This induction in response to ethylene was also observed at the translational level using antibodies to TR-ACO2. Analysis, in silico, of the 5′ flanking sequences to identify putative transcriptional binding domains reveals that both the TR-ACO2 and TR-ACO3 5′ flanking sequence contain domains that are associated with an ethylene-response. The significance of these observations in terms of how the expression of these ACO genes is regulated during leaf ontogeny in vivo is discussed.

Solubility and binding properties of PEGylated lysozyme derivatives with increasing molecular weight on hydrophobic-interaction chromatographic resins

Dynamic binding capacities and resolution of PEGylated lysozyme derivatives with varying molecular weights of poly (ethylene) glycol (PEG) with 5 kDa, 10 kDa and 30 kDa for HIC resins and columns are presented. To find the optimal range for the operating conditions, solubility studies were performed by high-throughput analyses in a 96-well plate format, and optimal salt concentrations and pH values were determined. The solubility of PEG-proteins was strongly influenced by the length of the PEG moiety. Large differences in the solubilities of PEGylated lysozymes in two different salts, ammonium sulfate and sodium chloride were found. Solubility of PEGylated lysozyme derivatives in ammonium sulfate decreases with increased length of attached PEG chains. In sodium chloride all PEGylated lysozyme derivatives are fully soluble in a concentration range between 0.1 mg protein/ml and 10 mg protein/ml. The binding capacities for PEGylated lysozyme to HIC resins are dependent on the salt type and molecular weight of the PEG polymer. In both salt solutions, ammonium sulfate and sodium chloride, the highest binding capacity of the resin was found for 5 kDa PEGylated lysozyme. For both native lysozyme and 30 kDa mono-PEGylated lysozyme the binding capacities were lower. In separation experiments on a TSKgel Butyl-NPR hydrophobic-interaction column with ammonium sulfate as mobile phase, the elution order was: native lysozyme, 5 kDa mono-PEGylated lysozyme and oligo-PEGylated lysozyme. This elution order was found to be reversed when sodium chloride was used. Furthermore, the resolution of the three mono-PEGylated forms was not possible with this column and ammonium sulfate as mobile phase. In 4 M sodium chloride a resolution of all PEGylated lysozyme forms was achieved. A tentative explanation for these phenomena can be the increased solvation of the PEG polymers in sodium chloride which changes the usual attractive hydrophobic forces in ammonium sulfate to more repulsive hydration forces in this hydrotrophic salt.

Lipoteichoic acid from Staphylococcus aureus attenuates differentiation of pre-osteoclast into mature osteoclast (136.26)

Abstract Bacterial virulence factors regulate differentiation and functions of osteoclasts, which mediate the bone-resorptive process. Here, we investigated the effect of a major cell wall virulence factor of Gram-positive bacteria, lipoteichoic acid (LTA), on the osteoclast differentiation. Highly-pure and structurally-intact LTA was prepared from Staphylococcus aureus through sequential application of organic solvent extraction and hydrophobic-interaction column chromatography followed by ion-exchange column chromatography. Osteoclast precursors were prepared from C57BL/6 mouse bone marrow cells using M-CSF and RANKL. Staphylococcal LTA suppressed the differentiation of osteoclast precursors to mature osteoclasts concordantly with an inhibition of the bone-resorptive capacity. Meanwhile, LTA augmented the phagocytic and inflammatory potential of the pre-osteoclasts. The LTA inhibition of osteoclast differentiation appears to be mediated through Toll-like receptor 2 (TLR2), and interestingly both MyD88-dependent and -independent signaling pathways. Furthermore, LTA reduced phosphorylation of ERK and JNK during the osteoclast differentiation process followed by a decreased DNA-binding activity of AP-1. Conclusively, our results suggest that LTA from S. aureus attenuates the differentiation of pre-osteoclast to mature osteoclast through TLR2 with partial involvement of MyD88 signaling pathways.

Evaluation of the impact of lipid fouling during the chromatographic purification of virus‐like particles from Saccharomyces cerevisiae

AbstractBACKGROUND: Production of recombinant virus‐like particles (VLPs) in yeast expression systems for use as vaccines requires cell disruption and detergent‐mediated steps to liberate the product. Typically, these release high levels of cellular components such as lipids that foul chromatography columns. This study compares the impact of applying lipid‐rich and lipid‐depleted feedstocks to hydrophobic interaction chromatography columns to quantify the loss of performance caused by the presence of host lipids over a total of 40 operational cycles.RESULTS: VLP binding capacity in the lipid‐rich feed was significantly lower than for the lipid‐depleted feed, with greater than 24% of the lipids remaining in the column after each cycle. Triacylglycerol was found to be the major contaminant. The effectiveness of subsequent caustic clean‐in‐place was limited, resulting in column hydrophobicity increasing over repeated loading cycles. This improved the effective VLP binding capacity and affinity, but also made product elution more difficult, and recovery decreased by more than 70% over the 40 cycles.CONCLUSION: Host cell lipids cause major fouling problems during VLP purification. Instead of screening for better CIP conditions, priority should be given to identifying suitable upstream lipid removal strategies in order to maintain column performance and so yield more economically viable processes. Copyright © 2009 Society of Chemical Industry

DNA spike studies for demonstrating improved clearance on chromatographic media

DNA spike clearance methods were used to demonstrate improved clearance factors on anion exchange and hydrophobic interaction columns used in the production of human therapeutic proteins. DNA clearance at large-scale was first measured for a monoclonal antibody expressed in Chinese Hamster Ovary (CHO) cells and an antibody fragment expressed in Escherichia coli. Small-scale spike experiments were then performed on individual chromatographic steps using host-specific DNA paired with TaqMan PCR assay methods. This approach has advantages of improved specificity, sensitivity, cost and throughput compared to other types of spike clearance methods. The anion exchange column used in the monoclonal antibody process was shown to have very high capacity for CHO DNA, resulting in greater than 7.1 log reduction. The anion exchange and hydrophobic interaction columns used in the antibody fragment process were shown to have high E. coli DNA clearance capability, with greater than 5.1 and 5.3 logs clearance, respectively. Compared to the large-scale process, higher log reduction values were achieved in small-scale spike clearance studies by challenging the chromatographic steps with load DNA levels 2–5 logs higher than the large-scale process levels. Using highly specific and sensitive spike clearance methods, we demonstrated consistently high DNA clearance factors for each of the production processes that meet industry and regulatory standards for human therapeutics. The method is applicable to a broad range of industrial scale processes where demonstration of the robustness of DNA clearance is necessary to support development or licensure of biopharmaceutical products.

Effects of thermal heterogeneity in hydrophobic interaction chromatography

Manipulating temperature and salt concentration can have a powerful effect on the separation effectiveness in hydrophobic interaction chromatography (HIC). However, use of temperature as an operating variable in large-scale applications may involve undesirable consequences such as radial heterogeneity of the column temperature. In this study non-ideal effects of heat transfer in HIC columns were analyzed. The radial temperature gradients were measured by thermocouples immersed in a bed packed into a preparative column. The column wall was either thermostatted by a water jacket or left under ambient conditions. The influence of ineffective column thermostatting and of heat losses on the radial temperature profiles was demonstrated and predicted by a model of heat dispersion in a packed bed. To analyze possible positive or negative effects of thermal heterogeneity on band propagation, non-isothermal chromatographic elution of a model protein (α-chymotrypsinogen A) was recorded under salt gradient conditions as well as at constant salt concentration. To predict temperature and concentration profiles a model of the column dynamics was used. The model accounted for kinetics of mass and heat transfer. A good agreement between experimental and simulated profiles was achieved. It was shown that by proper selection of the process conditions undesirable temperature effects can be avoided or controlled.

Branched‐chain α‐ketoacid dehydrogenase complex (BCKDC): purification from rat liver and inhibition by thiamin pyrophosphate (TPP)

BCKDC is a rate‐limiting enzyme in the branched‐chain amino acid catabolic pathway. This enzyme complex is regulated by a phosphorylation/dephosphorylation cycle. The specific kinase (branched‐ chain α‐ketoacid dehydrogenase kinase (BDK)) is responsible for inactivation of the complex by phosphorylation. We have developed the method for BCKDC purification from rat liver using hydrophobic interaction column chromatography (Shimomura et al., Arch. Biochem. Biophy., 283, 293‐299 (1990)). We here report the modification of the purification method to obtain the highly purified BCKDC associated with BDK. Using the purified enzyme, we analyzed the inhibition of BDK by TPP at two potassium ion (K+) concentrations. IC50 values of 4.6 and 8.0 µM and inhibition constant values of 3.2 and 16.4 µM were obtained in the presence of 20 and 100 mM K+, respectively. These results suggest that BDK is less sensitive to TPP inhibition under physiological TPP and K+ concentrations.

Modified Method for Purifying Rat Liver Branched-Chain α-Ketoacid Dehydrogenase Complex

Branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) is a rate-limiting enzyme in the branched-chain amino acid catabolic pathway. We have developed a method of BCKDC purification from rat liver using hydrophobic interaction column chromatography (Shimomura et al., Arch. Biochem. Biophys., 283, 293-299 (1990)). Here we report a modification of the method designed to obtain the purified enzyme with high reproducibility.

Purification and Biochemical Characterization of a Specific β-Glucosidase from the Digestive Fluid of Larvae of the Palm Weevil,Rhynchophorus palmarum

A β-glucosidase was purified from the digestive fluid of the palm weevil Rhynchophorus palmarum L. (Coleoptera: Curculionidae) by chromatography on anion-exchange, gel filtration, and hydrophobic interaction columns. The preparation was shown to be homogeneous on polyacrylamide gels, β-glucosidase is a monomeric protein with a molecular weight of 58 kDa based on its mobility in SDS-PAGE and 60 kDa based on gel filtration. Maximal β-glucosidase activity occurred at 55°C and pH 5.0. The purified β-glucosidase was stable at 37°C and its pH stability was in the range of 5.0–6.0. The enzyme readily hydrolyzed p-nitrophenyl-β-D-glucoside, cellobiose, cellodextrins and required strictly β-gluco configuration for activity. It cleaved glucose-glucose beta-(1–4) linkages better than β-(1–2), β-(1–3) and β-(1–6) linkages. The catalytic efficiency (Kcat/KM) values for p-nitrophenyl-β-D-glucoside and cellobiose were respectively 240.48 mM-1s-1 and 134.80 mM-1s-1. Beta-glucosidase was capable of catalysing transglucosylation reactions. The yield of glucosylation of 2-phenylethanol (20 %), catalysed by the beta-glucosidase in the presence of cellobiose as glucosyl donor, is lower than those reported previously with conventional sources of beta-glucosidases. In addition, the optimum pH is different for the hydrolysis (pH 5.0) and transglucosylation reactions (pH 6.6).

Purification and characterization of an extreme halothermophilic protease from a halophilic bacterium Chromohalobacter sp. TVSP101

An extreme halophilic bacterium was isolated from solar saltern samples and identified based on biochemical tests and 16S r RNA sequencing as Chromohalobacter sp. strain TVSP101. The halophilic protease was purified using ultrafiltration, ethanol precipitation, hydrophobic interaction column chromatography and gel permeation chromatography to 180 fold with 22% yield. The molecular mass of the protease determined by SDS PAGE was 66 kDa. The purified enzyme was salt dependent for its activity and stability with an optimum of 4.5 M NaCl. The optimum temperature for maximum protease activity was 75ºC. The protease was optimally active at pH 8 and retained more than 80% of its activity in the range of pH 7-10. Sucrose and glycine at 10% (w/v) were the most effective osmolytes, retained 100% activity in the absence of NaCl. The activity was completely inhibited by ZnCl2 (2 mM), 0.1% SDS and PMSF (1mM). The enzyme was not inhibited by 1mM of pepstatin, EDTA and PCMB. The protease was active and retained 100% it activity in 10% (v/v) DMSO, DMF, ethanol and acetone.

Structural and Biochemical Characterization of the Oxidoreductase NmDsbA3 from Neisseria meningitidis

DsbA is an enzyme found in the periplasm of Gram-negative bacteria that catalyzes the formation of disulfide bonds in a diverse array of protein substrates, many of which are involved in bacterial pathogenesis. Although most bacteria possess only a single essential DsbA, Neisseria meningitidis is unusual in that it possesses three DsbAs, although the reason for this additional redundancy is unclear. Two of these N. meningitidis enzymes (NmDsbA1 and NmDsbA2) play an important role in meningococcal attachment to human epithelial cells, whereas NmDsbA3 is considered to have a narrow substrate repertoire. To begin to address the role of DsbAs in the pathogenesis of N. meningitidis, we have determined the structure of NmDsbA3 to 2.3-A resolution. Although the sequence identity between NmDsbA3 and other DsbAs is low, the NmDsbA3 structure adopted a DsbA-like fold. Consistent with this finding, we demonstrated that NmDsbA3 acts as a thiol-disulfide oxidoreductase in vitro and is reoxidized by Escherichia coli DsbB (EcDsbB). However, pronounced differences in the structures between DsbA3 and EcDsbA, which are clustered around the active site of the enzyme, suggested a structural basis for the unusual substrate specificity that is observed for NmDsbA3.

Integrated process for the purification of antibodies combining aqueous two-phase extraction, hydrophobic interaction chromatography and size-exclusion chromatography

We have evaluated a process incorporating aqueous two-phase extraction, hydrophobic interaction chromatography (HIC) and size-exclusion chromatography (SEC) for the purification of human immunoglobulin G (IgG) from a Chinese hamster ovary (CHO) cell supernatant. These unit operations were chosen not only for allowing the removal of target impurities but also for facilitating the integration of different process units without the need for any conditioning step. Extraction in aqueous two-phase systems (ATPSs), composed of polyethylene glycol (PEG) and sodium citrate, allowed the concentration of the antibodies in the citrate-rich phase and the removal of the most hydrophobic compounds in the PEG-rich phase. An ATPS composed of 10% (w/w) PEG 3350 and 12% (w/w) citrate, at pH 6, allowed the recovery of IgG with a 97% yield, 41% HPLC purity and 72% protein purity. This bottom phase was then directly loaded on a phenyl-Sepharose HIC column. This intermediate purification step allowed the capture of the antibodies using a citrate mobile phase with 99% of the antibody recovered in the elution fractions, with 86% HPLC purity and 91% protein purity. Finally, SEC allowed the final polishing by removing IgG aggregates. HIC-eluted fractions were directly injected in a Superose 6 size-exclusion column affording a 100% pure IgG solution with 90% yield.

Molecular Basis of Prodrug Activation by Human Valacyclovirase, an α-Amino Acid Ester Hydrolase

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of alpha-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar to tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific alpha-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.

Biochemical characterization of a strictly specific beta‐galactosidase from the digestive juice of the palm weevil Rhynchophorus palmarum larvae

AbstractA beta‐galactosidase from the digestive juice of the palm weevil Rhynchophorus palmarum L. larvae was purified by chromatography on ion exchange, gel filtration, and hydrophobic interaction columns. The preparation was shown to be homogeneous on polyacrylamide gel. Beta‐galactosidase was a monomeric protein with a molecular weight of 62 kDa based on its mobility in sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 60 kDa based on gel filtration. Maximal enzyme activity occurred at 55°C and pH 5.0. The purified beta‐galactosidase was stable at 37°C and its pH stability was in the range of 4.6–6.0. Beta‐galactosidase was highly specific for the beta‐d‐galactosyl residue and beta‐(1‐4) linkage. The catalytic efficiency (Vmax/Km) values for p‐nitrophenyl‐beta‐d‐galactopyranoside, beta‐d‐galactosyl(1‐4)‐d‐glucose (lactose), beta‐d‐galactosyl(1‐4)‐d‐galactose and beta‐d‐galactosyl(1‐4)‐beta‐d‐galactosyl(1‐4)‐d‐glucose were, respectively, 72.95, 10.97, 20.74 and 12.73. 5,5‐Dithio‐bis(2‐nitrobenzoate) and sodium dodecyl sulfate inhibited completely the beta‐galactosidase activity. The enzyme was capable of catalyzing transgalactosylation reactions. The yield of galactosylation of 2‐phenylethanol (43%), catalyzed by the beta‐galactosidase in the presence of lactose as galactosyl donor, is higher than those reported previously with conventional sources of beta‐galactosidases. In addition, the optimum pH is different for the hydrolysis (pH 5.0) and transgalactosylation reactions (pH 6.0).

Regulation of UDP-glucose pyrophosphorylase isozyme UGP5 associated with cold-sweetening resistance in potatoes

The regulation of UDP-Glc pyrophosphorylase (UGPase) isozyme, UGP5, was investigated in potato tuber. The cDNA for UGP5 was cloned into the bacterial expression vector pET21d and recombinant (RC) enzyme was expressed in E. coli (BL21 star cells). The RC-UGP5 isozyme was purified to near homogeneity using salt precipitation, hydrophobic interaction, and anion-exchange column chromatography. Kinetic analysis revealed that in the synthesis direction, K(m) values for Glc-1-P (0.83 mM) and UTP (0.22 mM) were similar to those observed previously with the mother tuber (MT)-UGP5. In the pyrophosphorolysis direction, the K(m) values for UDP-Glc (0.68 mM) and PPi (0.56 mM) were slightly higher than those observed previously. Maximum reaction velocities (V(max)) for RC-UGP5 were also elevated. Since the molecular mass, charge, and amino acid sequence of the MT- and RC-UGP5 isozymes were identical, it was assumed that altered kinetic constants may be due to an improper folding of RC-UGP5 polypeptide. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and proteomic analysis demonstrated that the UGP5 isozyme was a single polypeptide with a calculated molecular mass of 51.8kDa consisting of 477 amino acids. Native PAGE and kinetic analysis revealed that this polypeptide was monomeric in nature. Immunoblotting with specific antibodies and LC-MS/MS data indicated that UGP5 did not require any post-translational modification (e.g., phosphorylation, O-glycosylation, oligomerization/de-oligomerization, or the presence of the regulatory 14-3-3 proteins) for its regulation. Additionally, the two closely associated isozymes UGP5 and UGP6 in the cv. Snowden are likely the result of allelic differences of UGPase at a single locus.

Toward chromatographic analysis of interacting protein networks

Protein complexes, collectively referred to as the cellular interactome, appear to play a major role in cellular regulation. At present it is thought that the interactome could be composed of hundreds of protein assemblies. The objective of the work described here was to examine the prospect that chromatographic methods widely used in the preparative isolation of native proteins could be incorporated into global proteomics methods in such a way that the primary structure of protein complexes of sufficient stability to survive chromatography could be recognized along with their participation in protein complexes. Because wide differences in sizes are a unique feature of protein complexes, size-exclusion chromatography (SEC) was incorporated into all the fractionation strategies examined. Anion-exchange chromatography (AEC) and hydrophobic-interaction chromatography (HIC) were also examined because of the broad utility that these methods have shown in the preparation of proteins with native structure. Slightly more than a third of all proteins identified in yeast lysates were found to elute from SEC, AEC, and HIC columns with an apparent molecular weight much higher than that predicted from their parent gene. These results were interpreted to mean that these proteins were migrating through columns as components of protein complexes. Based on studies with multidimensional SEC → RPLC (reversed-phase liquid chromatography), AEC → SEC, and HIC → SEC systems, it was concluded that recognition of proteins in complexes could be easily incorporated into multidimensional chromatographic methods for global proteomics when at least one of the fractionation dimensions included SEC of native proteins.