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Test Proteins (test + protein)
Selected AbstractsConfocal imaging of chromatographic fouling under flow conditionsJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 10 2007Sun Chau Siu Abstract BACKGROUND: The fouling impact of selected fouling species was assessed by utilising confocal scanning laser microscopy (CSLM) to image a packed chromatographic bed during operation. A custom-made flow cell was packed with Q Sepharose FF and loaded with partially clarified E. coli homogenate. Selective, multicoloured fluorescent dyes were used to label a bovine serum albumin (BSA) test protein (Cy5.5), dsDNA (PicoGreen) and host cell proteins (HCPs) (Cy3). The fouling caused by the various fluorescently labelled components was visualised as a result of the fluorescence emitted by the PicoGreen-labelled dsDNA and the Cy3-labelled protein in the foulant stream, and by testing the adsorptive capacity of a test protein (BSA) onto the resin prior to and post-fouling as well as following the application of a common CIP procedure. RESULTS: Values for the effective diffusivity of BSA (De) were derived from the confocal images and the fouling impact was assessed by comparing De values obtained from different fouling scenarios. Under the most extreme conditions examined, fouling caused a 20% reduction in capacity compared to a fresh bed. BSA diffusivity did not appear to be affected by the fouling conditions studied. Sequential CIP using 15 CVs of 1 mol L,1 NaCl then 15 CVs of 1 mol L,1 NaOH was shown to be effective in removing nucleic acids and HCPs. Subsequent BSA adsorption showed that the CIP regime successfully restored the column capacity to its original value. In contrast, 15 CVs of 1 mol L,1 NaCl were ineffective in removing dsDNA but substantially removed HCPs. CONCLUSION: CSLM was demonstrated to be a useful tool for visualising fouling mechanisms. Comparing the results obtained by this technique using different modes of chromatographic operation provided insights into the fouling characteristics of finite baths versus packed beds. Copyright © 2007 Society of Chemical Industry [source] Comparison of conventional FASTA identity searches with the 80 amino acid sliding window FASTA search for the elucidation of potential identities to known allergensMOLECULAR NUTRITION & FOOD RESEARCH (FORMERLY NAHRUNG/FOOD), Issue 8 2007Gregory S. Ladics Abstract Food and Agriculture Organization/World Health Organization (FAO/WHO) recommended that IgE cross-reactivity between a transgenic protein and allergen be considered when there is ,F 35% identity over a sliding "window" of 80 amino acids. Our objective was to evaluate the false positive and negative rates observed using the FAO/WHO versus conventional FASTA analyses. Data used as queries against allergen databases and analyzed to assess false positive rates included: 1102 hypothetical corn ORFs; 907 randomly selected proteins; 89 randomly selected corn proteins; and 97 corn seed proteins. To evaluate false negative rates of both methods: Bet v 1a along with several crossreacting fruit/vegetable allergens and a bean ,-amylase inhibitor were used as queries. Both methods were also evaluated for their ability to detect a putative nonallergenic test protein containing a sequence derived from Ara h 1. FASTA versions 3.3t0 and 3.4t25 were utilized. Data indicate a conventional FASTA analysis produced fewer false positives and equivalent false negative rates. Conventional FASTA versus sliding window derived E scores were generally more significant. Results suggest a conventional FASTA search provides more relevant identity to the query protein and better reflects the functional similarities between proteins. It is recommended that the conventional FASTA analysis be conducted to compare identities of proteins to allergens. [source] Stabilization of proteins by low molecular weight multi-ionsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 10 2002Donald S. Maclean Abstract A method is described to identify small molecule ligands that stabilize proteins. The procedure is based on the hypothesis that molecules of various sizes containing two to four charges should occasionally bind to unpaired charged sites on the surface of proteins and by crosslinking such residues stabilize the native state of the liganded protein. A simple turbidity assay is employed that detects inhibition of protein aggregation under selected sets of conditions. Eight test proteins were screened and in all cases specific ligands were identified that inhibited protein aggregation at millimolar to micromolar concentrations. Only small effects of these stabilizers on protein biological activities were found. In some, but not all cases, circular dichroism and fluorescence studies provided direct evidence of the binding of stabilizing ligands to the proteins suggesting multiple mechanisms of stabilization. This approach should be applicable to the development of excipients for the stabilization of pharmaceutical proteins and industrial enzymes as well as serve as starting points for second-generation inhibitors of increased affinity and specificity. © 2002 Wiley-Liss Inc. and the American Pharmaceutical Association J Pharm Sci 91:2220,2229, 2002 [source] New ,-amino phenylalanine tetrazole ligand for immobilized metal affinity chromatography of proteinsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 16-17 2008Genhu Lei Abstract A new chelating compound has been developed for use in the immobilized metal affinity chromatographic (IMAC) separation of proteins. The bidentate ligand, ,-amino phenylalanine tetrazole, 4, was synthesized via a five-step synthesis from N -fluorenylmethoxycarbonyl phenylalanine and then immobilized onto silica through the epoxide coupling procedure. The binding behavior of the resulting IMAC sorbent, following chelation with Zn2+ to a density of 183 ,mol Zn2+ ions/g silica, was characterized by the retention of proteins in the pH range of 5.0,8.0, and by the adsorption behavior of lysozyme with frontal chromatography at pH 6.0 and 8.0. The prepared column showed the separation ability to four test proteins and the retention time of these proteins increased with an increase in pH. From the derived isotherms, the adsorption capacity, qm, for the binding of lysozyme to immobilized Zn2+ -,-amino phenylalanine tetrazole,silica was found to be 1.21 ,mol/g at pH 6.0 and 1.20 ,mol/g sorbent at pH 8.0, respectively, whilst the dissociation constants KD at these pH values were 5.22×10,6 and 3.49×10,6 M, respectively, indicating that the lysozyme was retained more stable under alkaline conditions, although the binding capacity in terms of micromole protein per gram sorbent remained essentially unchanged. [source] Rapid Matrix-Assisted Refolding of Histidine-Tagged ProteinsCHEMBIOCHEM, Issue 5 2009Tetyana Dashivets Abstract Matrix refolded: The formation of inclusion bodies, which are amorphous aggregates of misfolded insoluble protein, during recombinant protein expression, is one of the biggest bottlenecks in protein science. We report a stepwise, rational optimization procedure for refolding of insoluble proteins (see scheme). In comparison to refolding in-solution, this parallelized, matrix-assisted approach allows the refolding of various proteins in a fast and efficient manner. The formation of inclusion bodies (IBs),amorphous aggregates of misfolded insoluble protein,during recombinant protein expression, is still one of the biggest bottlenecks in protein science. We have developed and analyzed a rapid parallel approach for matrix-assisted refolding of recombinant His6 -tagged proteins. Efficiencies of matrix-assisted refolding were screened in a 96-well format. The developed methodology allowed the efficient refolding of five different test proteins, including monomeric and oligomeric proteins. Compared to refolding in-solution, the matrix-assisted refolding strategy proved equal or better for all five proteins tested. Interestingly, specifically oligomeric proteins displayed significantly higher levels of refolding compared to refolding in-solution. Mechanistically, matrix-assisted folding seems to differ from folding in-solution, as the reaction proceeds more rapidly and shows a remarkably different concentration dependence,it allows refolding at up to 1000-fold higher protein concentration than folding in-solution. [source] | ||||||||||