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Protein G (protein + g)
Selected AbstractsMicro freef-low IEF enhanced by active cooling and functionalized gelsELECTROPHORESIS, Issue 24 2006Jacob W. Albrecht Abstract Rapid free-flow IEF is achieved in a microfluidic device by separating the electrodes from the focusing region with porous buffer regions. Moving the electrodes outside enables the use of large electric fields without the detrimental effects of bubble formation in the active region. The anode and cathode porous buffer regions, which are formed by acrylamide functionalized with immobilized pH groups, allow ion transport while providing buffering capacity. Thermoelectric cooling mitigates the effects of Joule heating on sample focusing at high field strengths (,500,V/cm). This localized cooling was observed to increase device performance. Rapid focusing of low-molecular-weight pI markers and Protein G,mouse IgG complexes demonstrate the versatility of the technique. Simulations provide insight into and predict device performance based on a well-defined sample composition. [source] Binding site on human immunoglobulin G for the affinity ligand HWRGWVJOURNAL OF MOLECULAR RECOGNITION, Issue 3 2010Haiou Yang Abstract Affinity ligand HWRGWV has demonstrated the ability to isolate human immunoglobulin G (hIgG) from mammalian cell culture media. The ligand specifically binds hIgG through its Fc portion. This work shows that deglycosylation of hIgG has no influence on its binding to the HWRGWV ligand and the ligand does not compete with Protein A or Protein G in binding hIgG. It is suggested by the mass spectrometry (MS) data and docking simulation that HWRGWV binds to the pFc portion of hIgG and interacts with the amino acids in the loop Ser383,Asn389 (SNGQPEN) located in the CH3 domain. Subsequent modeling has suggested a possible three-dimensional minimized solution structure for the interaction of hIgG and the HWRGWV ligand. The results support the fact that a peptide as small as a hexamer can have specific interactions with large proteins such as hIgG. Copyright © 2009 John Wiley & Sons, Ltd. [source] CE-based noncompetitive immunoassay for immunoglobulin G in bovine colostrum productsELECTROPHORESIS, Issue 21 2007Jin Zhao Abstract A CE-based noncompetitive immunoassay for IgG in bovine colostrum products was established. FITC-labeled protein G (FITC-PrG) was tagged through noncovalent bindings to the Fc region of the mouse monoclonal antibovine IgG (Ab). The FITC-PrG, Ab, and IgG formed a sandwiched immunocomplex FITC-PrG-Ab-IgG under optimal incubation conditions. The immunocomplex was separated and analyzed by CZE with LIF detection in less than 2,min in an uncoated fused-silica capillary. Addition of PEG 20,000 (PEG 20M) in the running buffer significantly suppressed analyte adsorption and thus improved the reproducibility and the resolution. The precision of the method was 5.1% (n,=,7). A linear relationship was established for the IgG concentration in the range of 1,5,mg/L with a linear correlation coefficient (r,=,0.9917). The LOD was 0.1,mg/L (S/N,=,3). The method was successfully applied for the determination of IgG in bovine colostrum products and satisfactory results were achieved. [source] Identifying native-like protein structures using physics-based potentialsJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 1 2002Brian N. Dominy Abstract As the field of structural genomics matures, new methods will be required that can accurately and rapidly distinguish reliable structure predictions from those that are more dubious. We present a method based on the CHARMM gas phase implicit hydrogen force field in conjunction with a generalized Born implicit solvation term that allows one to make such discrimination. We begin by analyzing pairs of threaded structures from the EMBL database, and find that it is possible to identify the misfolded structures with over 90% accuracy. Further, we find that misfolded states are generally favored by the solvation term due to the mispairing of favorable intramolecular ionic contacts. We also examine 29 sets of 29 misfolded globin sequences from Levitt's "Decoys ,R' Us" database generated using a sequence homology-based method. Again, we find that discrimination is possible with approximately 90% accuracy. Also, even in these less distorted structures, mispairing of ionic contacts results in a more favorable solvation energy for misfolded states. This is also found to be the case for collapsed, partially folded conformations of CspA and protein G taken from folding free energy calculations. We also find that the inclusion of the generalized Born solvation term, in postprocess energy evaluation, improves the correlation between structural similarity and energy in the globin database. This significantly improves the reliability of the hypothesis that more energetically favorable structures are also more similar to the native conformation. Additionally, we examine seven extensive collections of misfolded structures created by Park and Levitt using a four-state reduced model also contained in the "Decoys ,R' Us" database. Results from these large databases confirm those obtained in the EMBL and misfolded globin databases concerning predictive accuracy, the energetic advantage of misfolded proteins regarding the solvation component, and the improved correlation between energy and structural similarity due to implicit solvation. Z-scores computed for these databases are improved by including the generalized Born implicit solvation term, and are found to be comparable to trained and knowledge-based scoring functions. Finally, we briefly explore the dynamic behavior of a misfolded protein relative to properly folded conformations. We demonstrate that the misfolded conformation diverges quickly from its initial structure while the properly folded states remain stable. Proteins in this study are shown to be more stable than their misfolded counterparts and readily identified based on energetic as well as dynamic criteria. In summary, we demonstrate the utility of physics-based force fields in identifying native-like conformations in a variety of preconstructed structural databases. The details of this discrimination are shown to be dependent on the construction of the structural database. © 2002 Wiley Periodicals, Inc. J Comput Chem 23: 147,160, 2002 [source] Direct measurement of the transverse and longitudinal 15N chemical shift anisotropy,dipolar cross-correlation rate constants using 1H-coupled HSQC spectraMAGNETIC RESONANCE IN CHEMISTRY, Issue 10 2003Jennifer B. Hall Abstract We describe direct methods for the measurement of the transverse and longitudinal 15N chemical shift anisotropy,dipolar cross-correlation rates based on comparison of the 15N doublet components observed in 1H-coupled 1H,15N HSQC-type spectra. This allows the determination of the cross-correlation rates with no need for correction factors associated with other methods. The signal overlap problem of coupled HSQC spectra is addressed by using the IPAP scheme (Ottiger M, Delaglio F, Bax A. J. Magn. Reson. 1998; 131: 373). The methods proposed here use a conventional t1 evolution period, which allows one to minimize the truncation artifacts observed in a constant-time-type experiment (Hall JB, Dayie K, Fushman D. J. Biomol. NMR 2003; 26: 181). Applications of these measurements to the B3 domain of protein G are discussed. Copyright © 2003 John Wiley & Sons, Ltd. [source] Intermediates and the folding of proteins L and GPROTEIN SCIENCE, Issue 4 2004Scott Brown Abstract We use a minimalist protein model, in combination with a sequence design strategy, to determine differences in primary structure for proteins L and G, which are responsible for the two proteins folding through distinctly different folding mechanisms. We find that the folding of proteins L and G are consistent with a nucleation-condensation mechanism, each of which is described as helix-assisted ,-1 and ,-2 hairpin formation, respectively. We determine that the model for protein G exhibits an early intermediate that precedes the rate-limiting barrier of folding, and which draws together misaligned secondary structure elements that are stabilized by hydrophobic core contacts involving the third ,-strand, and presages the later transition state in which the correct strand alignment of these same secondary structure elements is restored. Finally, the validity of the targeted intermediate ensemble for protein G was analyzed by fitting the kinetic data to a two-step first-order reversible reaction, proving that protein G folding involves an on-pathway early intermediate, and should be populated and therefore observable by experiment. [source] Designed protein G core variants fold to native-like structures: Sequence selection by ORBIT tolerates variation in backbone specificationPROTEIN SCIENCE, Issue 2 2001Scott A. Ross Abstract The solution structures of two computationally designed core variants of the ,1 domain of streptococcal protein G (G,1) were solved by 1H NMR methods to assess the robustness of amino acid sequence selection by the ORBIT protein design package under changes in protein backbone specification. One variant has mutations at three of 10 core positions and corresponds to minimal perturbations of the native G,1 backbone. The other, with mutations at six of 10 positions, was calculated for a backbone in which the separation between G,1's ,-helix and ,-sheet was increased by 15% relative to native G,1. Exchange broadening of some resonances and the complete absence of others in spectra of the sixfold mutant bespeak conformational heterogeneity in this protein. The NMR data were sufficiently abundant, however, to generate structures of similar, moderately high quality for both variants. Both proteins adopt backbone structures similar to their target folds. Moreover, the sequence selection algorithm successfully predicted all core ,1 angles in both variants, five of six ,2 angles in the threefold mutant and four of seven ,2 angles in the sixfold mutant. We conclude that ORBIT calculates sequences that fold specifically to a geometry close to the template, even when the template is moderately perturbed relative to a naturally occurring structure. There are apparently limits to the size of acceptable perturbations: In this study, the larger perturbation led to undesired dynamic behavior. [source] Effects of segment substitution on the structure and stability of immunoglobulin G binding domain of streptococcal protein GBIOPOLYMERS, Issue 1 2005Hai-Ning Du Abstract Structural formation of segments plays pivotal roles in protein folding and stability, but how the segment influences the structural ensemble remains elusive. We engineered two hybrid proteins by replacing the central helical segment of immunoglobulin G binding domain of streptococcal protein G with an ,-helix or ,2 -strand element of a structural homologue, the immunoglobulin G binding domain of streptococcal protein L. The results show that substitution by the ,-helical sequence retains a folded structure predominantly with a three-stranded ,-sheet but slightly destabilizes the compact ensemble, while substitution by the ,2 -strand sequence completely destroys the structural formation. The finding implies that the local segment may influence the tertiary structure and overall stability, and the tertiary interactions may modulate structural formation of the segment, which might be considered when studying protein folding, prediction, design, and engineering. © 2005 Wiley Periodicals, Inc. Biopolymers 79: 9,17, 2005 This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] Binding and entry of respiratory syncytial virus into host cells and initiation of the innate immune responseCELLULAR MICROBIOLOGY, Issue 10 2003James Harris Summary Respiratory syncytial virus (RSV) is the most common cause of severe lower respiratory tract infection in infants and the elderly. There is currently no effective antiviral treatment for the infection, but advances in our understanding of RSV uptake, especially the role of surfactant proteins, the attachment protein G and the fusion protein F, as well as the post-binding events, have revealed potential targets for new therapies and vaccine development. RSV infection triggers an intense inflammatory response, mediated initially by the infected airway epithelial cells and antigen-presenting cells. Humoral and cell-mediated immune responses are important in controlling the extent of infection and promoting viral clearance. The initial innate immune response may play a critical role by influencing the subsequent adaptive response generated. This review summarizes our current understanding of RSV binding and uptake in mammalian cells and how these initial interactions influence the subsequent innate immune response generated. [source] Infectious gastroenteritis caused by Vibrio harveyi (V. carchariae) in cultured red drum, Sciaenops ocellatusJOURNAL OF APPLIED ICHTHYOLOGY, Issue 1 2003P.-C. Liu Summary An outbreak of serious mortality among the cultured red drum Sciaenops ocellatus (L.) characterized by a swollen intestine containing transparent yellow fluid (ascites and gastroenteritis) occurred in July 2000 in Taiwan. A motile strain Rd 0700 was isolated from head kidney and/or the intestinal yellow fluid on tryptone soya agar (TSA) supplemented with 2% (w/v) NaCl and/or thiosulfate citrate bile salt (TCBS) sucrose agar plates. Applying biochemical characteristics, this strain was characterized and identified as Vibrio harveyi (V. carchariae). The bacteria could be re-isolated from kidney, liver, and the transparent yellow fluid of swollen intestine of fish after bacterial challenge. The LD50 values of the organism and its extracellular products (ECP) were 2.9×107 colony forming units (CFU) and 3.85 ,g protein g,1 fish body weight, respectively. All moribund/dead fish exhibited gastroenteritis except those killed within 12 h. This is a first report showing that intraperitoneal (i.p.) injection of the ECP from V. carchariae is lethal to red drum and can reproduce gastroenteritis in the fish. [source] Imprinting Status of G,S, NESP55, and XL,s in Cell Cultures Derived from Human Embryonic Germ Cells: GNAS Imprinting in Human Embryonic Germ CellsCLINICAL AND TRANSLATIONAL SCIENCE, Issue 5 2009Janet L. Crane M.D. Abstract GNAS is a complex gene that through use of alternative first exons encodes signaling proteins G,s and XL,s plus neurosecretory protein NESP55. Tissue-specific expression of these proteins is regulated through reciprocal genomic imprinting in fully differentiated and developed tissue. Mutations in GNAS account for several human disorders, including McCune-Albright syndrome and Albright hereditary osteodystrophy, and further knowledge of GNAS imprinting may provide insights into variable phenotypes of these disorders. We therefore analyzed expression of G,s, NESP55, and XL,s prior to tissue differentiation in cell cultures derived from human primordia germ cells. We found that the expression of G,s was biallelic (maternal allele: 52.6%± 2.5%; paternal allele: 47.2%± 2.5%; p= 0.07), whereas NESP55 was expressed preferentially from the maternal allele (maternal allele: 81.9%± 10%; paternal allele: 18.1%± 10%; p= 0.002) and XL,s was preferentially expressed from the paternal allele (maternal allele: 2.7%± 0.3%; paternal allele: 97.3%± 0.3%; p= 0.007). These results demonstrate that imprinting of NESP55 occurs very early in development, although complete imprinting appears to take place later than 5,11 weeks postfertilization, and that imprinting of XL,s occurs very early postfertilization. By contrast, mprinting of G,s most likely occurs after 11 weeks postfertilization and after tissue differentiation. [source] | |||||||||||||