TY - JOUR TI - Effects of peptide density and elution pH on affinity chromatographic purification of human immunoglobulins A and M AU - Liu, Zhuo AU - Gurgel, Patrick V. AU - Carbonell, Ruben G. T2 - JOURNAL OF CHROMATOGRAPHY A AB - A family of linear hexamer peptide ligands HWRGWV, HYFKFD and HFRRHL, initially identified for their affinity to the Fc portion of human immunoglobulin G (hIgG), also have potential for use in the purification of human immunoglobulins A (hIgA) and M (hIgM). HWRGWV demonstrated the strongest binding affinity to hIgM, followed by hIgA and hIgG respectively. The effects of N-terminal acetylation of the peptide, as well as elution buffer pH, on the chromatographic elution of human IgG, IgA and IgM from HWRGWV resins at various peptide densities (0.04–0.55 meq/g) were investigated. Over 80% recovery and 90% purity were achieved for human IgG and IgA isolation from complete minimum essential medium (cMEM) using HWRGWV resin at optimum peptide densities. For human IgM, 75.7% recovery and 86.0% purity were achieved by using HWRGWV at a low peptide density of 0.04 meq/g. Although HYFKFD and HFRRHL exhibited their ability for isolation of human IgG, IgA and IgM from cMEM as well, HWRGWV is the best option among them for large-scale purification of human IgG, IgA and IgM based on conditions tested. DA - 2011/11/18/ PY - 2011/11/18/ DO - 10.1016/j.chroma.2011.09.038 VL - 1218 IS - 46 SP - 8344-8352 SN - 1873-3778 KW - Human immunoglobulin KW - IgG KW - IgA KW - IgM KW - Affinity chromatography KW - Binding site ER - TY - JOUR TI - Selectively solvated triblock copolymer networks under biaxial strain AU - Krishnan, Arjun S. AU - Zanten, John H. AU - Seifert, Soenke AU - Lee, Byeongdu AU - Spontak, Richard J. T2 - APPLIED PHYSICS LETTERS AB - Triblock copolymers swollen with a midblock-selective solvent provide a test platform by which to interrogate the properties of highly elastic physical gel networks. Here, such networks are biaxially strained and studied by synchrotron small-angle x-ray scattering. Analysis of the form factor reveals that initially spherical micellar cores deform to ellipsoids when strained. The Percus-Yevick hard-sphere model describes the structure factor of micelles exhibiting liquid-like order prior to deformation but requires an attractive potential to match the structure factor under strain. The magnitude of this potential increases with increasing strain, indicating a change in coronal overlap as the network is stretched. DA - 2011/9/5/ PY - 2011/9/5/ DO - 10.1063/1.3635780 VL - 99 IS - 10 SP - SN - 0003-6951 ER - TY - JOUR TI - Effects of UV Exposure and Initiator Concentration on the Spatial Variation of Poly(glycidyl methacrylate) Grafts on Nonwoven Fabrics AU - Zheng, Yong AU - Gurgel, Patrick V. AU - Carbonell, Ruben G. T2 - INDUSTRIAL & ENGINEERING CHEMISTRY RESEARCH AB - This paper describes the spatial uniformity of grafted layers of poly(glycydyl methacrylate) on the fibers of polypropylene nonwoven fabrics, and how they depend on the UV pretreatment step, the adsorption of initiator (benzophenone) to the fiber surface, and the UV grafting step. UV light transmission inside the nonwoven fabrics was measured to determine the effect of light intensity variations during the pretreatment and grafting steps. The light intensity decay depends on the pore size of the fabric and the presence of solvents in the nonwoven fabric. The adsorption of benzophenone to the fiber surface is lower in regions of low light intensity, resulting in a spatial variation due to the UV pretreatment. The presence of solvents tends to reduce light intensity decay during the grafting step. The results of this paper indicate how to avoid spatial nonuniformities during grafting by controlling the reaction conditions. DA - 2011/5/18/ PY - 2011/5/18/ DO - 10.1021/ie1021333 VL - 50 IS - 10 SP - 6115-6123 SN - 0888-5885 ER - TY - JOUR TI - Polystyrene/Decahydronaphthalene/Propane Phase Equilibria and Polymer Conformation Properties from Intrinsic Viscosities AU - Cain, Nathaniel AU - Haywood, Alexander AU - Roberts, George AU - Kiserow, Douglas AU - Carbonell, Ruben T2 - JOURNAL OF POLYMER SCIENCE PART B-POLYMER PHYSICS AB - Abstract The influence of dissolved propane (up to 31.2 wt %) on the phase equilibria of 5 wt % polystyrene (PS) dissolved in 66/34 wt % trans/cis ‐decahydronaphthalene (DHN) was measured over the temperature range of 323–423 K. A suitable temperature, pressure, and propane composition operating space was defined to measure intrinsic viscosities of a single fluid phase. Intrinsic viscosities of PS in cosolvent mixtures of propane and trans/cis ‐DHN were measured between 323 and 423 K and between 70 and 208 bar. The addition of propane to the isomeric mixture of DHN resulted in a decreased solvent quality for PS, causing a contraction of the PS coil. The most dramatic decrease in solvent quality with the addition of propane occurred at 323 K and 70 bar with approximately a 36% reduction in the viscometric radius with the addition of 45 mol % propane to DHN. At 423 K, the solvent quality was less sensitive to the addition of propane and only a 13% reduction in the viscometric radius was observed at 70 bar and 45 mol % propane in DHN. © 2011 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2011 DA - 2011/8/1/ PY - 2011/8/1/ DO - 10.1002/polb.22282 VL - 49 IS - 15 SP - 1093-1100 SN - 0887-6266 KW - conformational analysis KW - decahydronaphthalene KW - intrinsic viscosity KW - miscibility KW - phase behavior KW - phase diagrams KW - phase separation KW - polystyrene KW - viscosity ER - TY - JOUR TI - Performance of hexamer peptide ligands for affinity purification of immunoglobulin G from commercial cell culture media AU - Naik, A. D. AU - Menegatti, S. AU - Gurgel, P. V. AU - Carbonell, R. G. T2 - Journal of Chromatography A AB - Previous work has reported on the identification and characterization of the hexapeptide ligands HWRGWV, HYFKFD, and HFRRHL for the affinity capture of IgG through specific binding to its Fc fragment. This paper addresses issues related to the successful application of these ligands, on a commercial methacrylate chromatographic resin, for the purification of IgG from mammalian cell culture fluids. The concentrations of sodium chloride and sodium caprylate in the binding buffer were optimized to maximize the purity and yield of IgG upon elution. Screening of several regeneration conditions found that either 2 M guanidine–HCl or a combination of 0.85% phosphoric acid followed by 2 M urea resulted in complete recovery of the IgG adsorption capacity and that the column could be reused over many cycles. The hexapeptide ligands were used for the purification of humanized and chimeric monoclonal antibodies from two commercial CHO cell culture fluids. The chimeric MAb of IgG1 subclass was purified using the HWRGWV resin whereas the humanized MAb of IgG4 subclass was purified using the HWRGWV, HYFKFD and HFRRHL resins. The purities and yields obtained for both the MAbs were found to be higher than 94% and 85% respectively. These results compare well with the yields and purities obtained using Protein G columns. The residual DNA and host cell protein reduction obtained by the HWRGWV resin was in the range of 4 log reduction value (LRV) and 2 LRV respectively, comparable to those reported for Protein A resins. The dynamic binding capacity of all three peptide resins for the humanized monoclonal antibody was in the range of 20 mg/mL. DA - 2011/4/1/ PY - 2011/4/1/ DO - 10.1016/j.chroma.2010.11.071 VL - 1218 IS - 13 SP - 1691–1700 KW - Hexapeptide ligand KW - IgG purification KW - Monoclonal antibody KW - Affinity chromatography KW - Regeneration ER - TY - JOUR TI - Comparative Analysis of the 15.5kD Box C/D snoRNP Core Protein in the Primitive Eukaryote Giardia lamblia Reveals Unique Structural and Functional Features AU - Biswas, Shyamasri AU - Buhrman, Greg AU - Gagnon, Keith AU - Mattos, Carla AU - Brown, Bernard A., II AU - Maxwell, E. Stuart T2 - BIOCHEMISTRY AB - Box C/D ribonucleoproteins (RNP) guide the 2′-O-methylation of targeted nucleotides in archaeal and eukaryotic rRNAs. The archaeal L7Ae and eukaryotic 15.5kD box C/D RNP core protein homologues initiate RNP assembly by recognizing kink-turn (K-turn) motifs. The crystal structure of the 15.5kD core protein from the primitive eukaryote Giardia lamblia is described here to a resolution of 1.8 Å. The Giardia 15.5kD protein exhibits the typical α−β−α sandwich fold exhibited by both archaeal L7Ae and eukaryotic 15.5kD proteins. Characteristic of eukaryotic homologues, the Giardia 15.5kD protein binds the K-turn motif but not the variant K-loop motif. The highly conserved residues of loop 9, critical for RNA binding, also exhibit conformations similar to those of the human 15.5kD protein when bound to the K-turn motif. However, comparative sequence analysis indicated a distinct evolutionary position between Archaea and Eukarya. Indeed, assessment of the Giardia 15.5kD protein in denaturing experiments demonstrated an intermediate stability in protein structure when compared with that of the eukaryotic mouse 15.5kD and archaeal Methanocaldococcus jannaschii L7Ae proteins. Most notable was the ability of the Giardia 15.5kD protein to assemble in vitro a catalytically active chimeric box C/D RNP utilizing the archaeal M. jannaschii Nop56/58 and fibrillarin core proteins. In contrast, a catalytically competent chimeric RNP could not be assembled using the mouse 15.5kD protein. Collectively, these analyses suggest that the G. lamblia 15.5kD protein occupies a unique position in the evolution of this box C/D RNP core protein retaining structural and functional features characteristic of both archaeal L7Ae and higher eukaryotic 15.5kD homologues. DA - 2011/4/12/ PY - 2011/4/12/ DO - 10.1021/bi1020474 VL - 50 IS - 14 SP - 2907-2918 SN - 0006-2960 ER - TY - JOUR TI - Microviscoelasticity of soft repulsive sphere dispersions: Tracer particle microrheology of triblock copolymer micellar liquids and soft crystals AU - Tanner, Shaun A. AU - Amin, Samiul AU - Kloxin, Christopher J. AU - Zanten, John H. T2 - JOURNAL OF CHEMICAL PHYSICS AB - Tracer particle microrheology using diffusing wave spectroscopy-based microrheology is demonstrated to be a useful method to study the dynamics of aqueous Pluronic™ F108 solutions, which are viewed as solutions of repulsive soft spheres. The measured zero-shear microviscosity of noncrystallizing micellar dispersions indicates micelle corona dehydration upon increasing temperature. Colloidal sphere thermal motion is shown to be exquisitely sensitive to the onset of crystallization in these micellar dispersions. High temperature dynamics are dominated by an apparent soft repulsive micelle-micelle interaction potential indicating the important role played by lubrication forces and ultimately micelle corona interpenetration and compression at sufficiently high concentrations. The measured microscopic viscoelastic storage and loss moduli are qualitatively similar to those experimentally observed in mechanical measurements on colloidal dispersions and crystals, and calculated from mode coupling theory of colloidal suspensions. The observation of subdiffusive colloidal sphere thermal motion at short time-scales is strong evidence that the observed microscopic viscoelastic properties reflect the dynamics of individual micelles rather than a dispersion of micellar crystallites. DA - 2011/5/7/ PY - 2011/5/7/ DO - 10.1063/1.3578183 VL - 134 IS - 17 SP - SN - 1089-7690 ER - TY - JOUR TI - Modeling the thermodynamic and transport properties of decahydronaphthalene/propane mixtures: Phase equilibria, density, and viscosity AU - Cain, Nathaniel AU - Roberts, George AU - Kiserow, Douglas AU - Carbonell, Ruben T2 - FLUID PHASE EQUILIBRIA AB - Abstract The density and viscosity of propane mixed with 66/34 trans/cis -decahydronaphthalene were measured over a wide range of temperatures (323–423 K), pressures (2.5–208 bar), and compositions (0–65 mol% propane). For conditions giving two phases, the composition of the dense phase was measured in addition to the density and viscosity. The modified Sanchez-Lacombe Equation of State (MSLEOS) was used with a single linearly temperature-dependent pseudo-binary interaction parameter to correlate the phase compositions and densities. The compositions and densities of the mixtures were captured well with absolute average deviations between the model and the data of 5.3% and 2.3%, respectively. The mixture viscosities were computed from a free volume model (FVM) by using a single constant binary interaction parameter. Density predictions from the MSLEOS were used as input mixture density values required for the FVM. The FVM was found to correlate well with the mixture viscosity data with an absolute average deviation between the model and the data of 5.7%. DA - 2011/6/15/ PY - 2011/6/15/ DO - 10.1016/j.fluid.2011.02.009 VL - 305 IS - 1 SP - 25-33 SN - 0378-3812 KW - Phase equilibria KW - Modified Sanchez-Lacombe Equation of State KW - Decahydronaphthalene KW - Propane KW - density KW - Viscosity KW - Volume expansion KW - Viscosity reduction KW - Free volume model for viscosity ER - TY - JOUR TI - Functional Analysis of a Novel Motif Conserved across Geminivirus Rep Proteins AU - Nash, Tara E. AU - Dallas, Mary B. AU - Reyes, Maria Ines AU - Buhrman, Gregory K. AU - Ascencio-Ibanez, J. Trinidad AU - Hanley-Bowdoin, Linda T2 - JOURNAL OF VIROLOGY AB - ABSTRACT Members of the Geminiviridae have single-stranded DNA genomes that replicate in nuclei of infected plant cells. All geminiviruses encode a conserved protein (Rep) that catalyzes initiation of rolling-circle replication. Earlier studies showed that three conserved motifs—motifs I, II, and III—in the N termini of geminivirus Rep proteins are essential for function. In this study, we identified a fourth sequence, designated GRS ( g eminivirus R ep s equence), in the Rep N terminus that displays high amino acid sequence conservation across all geminivirus genera. Using the Rep protein of Tomato golden mosaic virus (TGMV AL1), we show that GRS mutants are not infectious in plants and do not support viral genome replication in tobacco protoplasts. GRS mutants are competent for protein-protein interactions and for both double- and single-stranded DNA binding, indicating that the mutations did not impair its global conformation. In contrast, GRS mutants are unable to specifically cleave single-stranded DNA, which is required to initiate rolling-circle replication. Interestingly, the Rep proteins of phytoplasmal and algal plasmids also contain GRS-related sequences. Modeling of the TGMV AL1 N terminus suggested that GRS mutations alter the relative positioning of motif II, which coordinates metal ions, and motif III, which contains the tyrosine involved in DNA cleavage. Together, these results established that the GRS is a conserved, essential motif characteristic of an ancient lineage of rolling-circle initiators and support the idea that geminiviruses may have evolved from plasmids associated with phytoplasma or algae. DA - 2011/2// PY - 2011/2// DO - 10.1128/jvi.02143-10 VL - 85 IS - 3 SP - 1182-1192 SN - 1098-5514 ER - TY - JOUR TI - Allosteric Modulation of Ras-GTP Is Linked to Signal Transduction through RAF Kinase AU - Buhrman, Greg AU - Kumar, V. S. Senthil AU - Cirit, Murat AU - Haugh, Jason M. AU - Mattos, Carla T2 - JOURNAL OF BIOLOGICAL CHEMISTRY AB - Ras is a key signal transduction protein in the cell. Mutants of Gly(12) and Gln(61) impair GTPase activity and are found prominently in cancers. In wild type Ras-GTP, an allosteric switch promotes disorder to order transition in switch II, placing Gln(61) in the active site. We show that the "on" and "off" conformations of the allosteric switch can also be attained in RasG12V and RasQ61L. Although both mutants have similarly impaired active sites in the on state, RasQ61L stabilizes an anti-catalytic conformation of switch II in the off state of the allosteric switch when bound to Raf. This translates into more potent activation of the MAPK pathway involving Ras, Raf kinase, MEK, and ERK (Ras/Raf/MEK/ERK) in cells transfected with RasQ61L relative to RasG12V. This differential is not observed in the Raf-independent pathway involving Ras, phosphoinositide 3-kinase (PI3K), and Akt (Ras/PI3K/Akt). Using a combination of structural analysis, hydrolysis rates, and experiments in NIH-3T3 cells, we link the allosteric switch to the control of signaling in the Ras/Raf/MEK/ERK pathway, supporting a GTPase-activating protein-independent model for duration of the Ras-Raf complex. DA - 2011/2/4/ PY - 2011/2/4/ DO - 10.1074/jbc.m110.193854 VL - 286 IS - 5 SP - 3323-3331 SN - 1083-351X ER - TY - JOUR TI - Transport and Binding Characterization of a Novel Hybrid Particle Impregnated Membrane Material for Bioseparations AU - Herigstad, M. Omon AU - Gurgel, Patrick V. AU - Carbonell, Ruben G. T2 - BIOTECHNOLOGY PROGRESS AB - Abstract The transport and binding properties of a novel hybrid particle‐nonwoven membrane medium are described. In this construct, a polymeric chromatographic resin is entrapped between two layers of a nonwoven polypropylene membrane. The membrane‐supported resin medium offers the advantage of increased interstitial pore diameter to allow passage of cells and other debris in the feed, while providing sufficiently high surface area for product capture within the resin particles. Columns packed with PIM displayed excellent flow distribution and had interstitial porosities of 0.48 ± 0.01, 25–60% larger than those typical of a packed bed. These columns were able to pass over 95% of E. coli cells and human red blood cell concentrate in 30 column volumes while maintaining a pressure drop significantly lower than that of a packed bed with a similar amount of resin. The dynamic binding capacity of bovine serum albumin (BSA) to the chromatographic resin entrapped in the PIM packed column was essentially the same as that observed with the same volume of resin in a packed bed. The General Rate (GR) model of chromatography was used to analyze experiments indicating the breakthrough behavior of the PIM columns is predictable, and very similar to those of a normal packed bed. These results suggest that PIM constructs can be designed to process viscous mobile phases containing particulates while retaining the desirable binding characteristics of the embedded chromatographic resin and could find uses in adsorption separation processes from complex feed streams such as whole blood, cell culture, and food processing. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2011 DA - 2011/// PY - 2011/// DO - 10.1002/btpr.502 VL - 27 IS - 1 SP - 129-139 SN - 1520-6033 KW - particle-impregnated membrane KW - process integration KW - mathematical modeling ER -