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Ribonuclease B (ribonuclease + b)

Distribution by Scientific Domains
Chemistry71%

Selected Abstracts

Arthrobacter Endo-,- N -Acetylglucosaminidase Shows Transglycosylation Activity on Complex-Type N -Glycan Oxazolines: One-Pot Conversion of Ribonuclease B to Sialylated Ribonuclease C

CHEMBIOCHEM, Issue 10 2010
Wei Huang Dr.
Transforming protein glycosylation: The Arthrobacter endo-,- N -acetylglucosaminidase (Endo-A) was found to transglycosylate complex N -glycan oxazolines without product hydrolysis. Given its high activity in hydrolyzing high-mannose-type N -glycans, the newly discovered property enables a one-pot switch of glycans in a glycoprotein, as shown by the conversion of heterogeneous RNase B to homogeneous sialylated RNase C. [source]

Microchip isoelectric focusing with monolithic immobilized pH gradient materials for proteins separation

ELECTROPHORESIS, Issue 23 2009
Yu Liang
Abstract Monolithic immobilized pH gradient (M-IPG) materials were prepared in microchannles by photoinitiated polymerization of acrylamide, glycidylmethacrylate and Bis, followed by the attachment of focused Ampholine onto the surface of porous monoliths via epoxide groups. With M-IPG materials as matrix, FITC-labeled ribonuclease B, myoglobin and ,-casein were well separated by microchip isoelectric focusing (,CIEF) without carrier amphocytes (CAs) added in the buffer. Both chemical and pressure mobilization were applied to drive focused zones for LIF detection. Our experimental results showed that pressure mobilization was preferable with neglectable band broadening, and good peak shape and high detection sensitivity were obtained. All these results demonstrate that ,CIEF with M-IPG materials is not only an efficient mode for protein enrichment and separation but also attractive to couple with other CE modes to achieve multi-dimensional separation or MS for further identification, without the interference of mobile CAs. [source]

Multilayer poly(vinyl alcohol)-adsorbed coating on poly(dimethylsiloxane) microfluidic chips for biopolymer separation

ELECTROPHORESIS, Issue 1 2005
Dapeng Wu
Abstract A poly(dimethylsiloxane) (PDMS) microfluidic chip surface was modified by multilayer-adsorbed and heat-immobilized poly(vinyl alcohol) (PVA) after oxygen plasma treatment. The reflection absorption infrared spectrum (RAIRS) showed that 88% hydrolyzed PVA adsorbed more strongly than 100% hydrolyzed one on the oxygen plasma-pretreated PDMS surface, and they all had little adsorption on original PDMS surface. Repeating the coating procedure three times was found to produce the most robust and effective coating. PVA coating converted the original PDMS surface from a hydrophobic one into a hydrophilic surface, and suppressed electroosmotic flow (EOF) in the range of pH 3,11. More than 1 000,000 plates/m and baseline resolution were obtained for separation of fluorescently labeled basic proteins (lysozyme, ribonuclease B). Fluorescently labeled acidic proteins (bovine serum albumin, ,-lactoglobulin) and fragments of dsDNA ,X174 RF/HaeIII were also separated satisfactorily in the three-layer 88% PVA-coated PDMS microchip. Good separation of basic proteins was obtained for about 70 consecutive runs. [source]

Development of fully functional proteins with novel glycosylation via enzymatic glycan trimming

JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 8 2009
Melinda L. Toumi
Abstract Recombinant glycoproteins present unique challenges to biopharmaceutical development, especially when efficacy is affected by glycosylation. In these cases, optimizing the protein's glycosylation is necessary, but difficult, since the glycan structures cannot be genetically encoded, and glycosylation in nonhuman cell lines can be very different from human glycosylation profiles. We are exploring a potential solution to this problem by designing enzymatic glycan optimization methods to produce proteins with useful glycan compositions. To demonstrate viability of this new approach to generating glycoprotein-based pharmaceuticals, the N -linked glycans of a model glycoprotein, ribonuclease B (RNase B), were modified using an ,-mannosidase to produce a new glycoprotein with different glycan structures. The secondary structure of the native and modified glycoproteins was retained, as monitored using circular dichroism. An assay was also developed using an RNA substrate to verify that RNase B had indeed retained its function after being subjected to the necessary glycan modification conditions. This is the first study that verifies both activity and secondary structure of a glycoprotein after enzymatic glycan trimming for use in biopharmaceutical development methods. The evidence of preserved structure and function for a modified glycoprotein indicates that extracellular enzymatic modification methods could be implemented in producing designer glycoproteins. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:2581,2591, 2009 [source]

Laser-improved protein crystallization screening

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 8 2010
Neela Yennawar
Screening of proteins for crystallization under laser irradiation was investigated using six proteins: ribonuclease B, glucose dehydrogenase, lysozyme, sorbitol dehydrogenase, fructose dehydrogenase and myoglobin. Shining 532,nm green circularly polarized laser light with a picosecond pulse and 6,mW power for 30,s on newly set-up protein drops showed a marked improvement in the number of screen conditions amenable for crystal growth compared with control drops under identical conditions but without laser exposure. For glucose dehydrogenase and sorbitol dehydrogenase, larger and better quality crystals were formed and the resolution of X-ray diffraction was improved. The speed of crystallization increased in the case of ribonuclease B, lysozyme and sorbitol dehydrogenase. During laser irradiation, the amount of precipitation in the screened drops increased, indicating a transient decrease in protein solubility. At the optimized laser settings, there was no deleterious effect of the laser on crystal growth or on the protein. In the cases of ribonuclease B and lysozyme the crystal packing did not change owing to the laser exposure. [source]

Rapid analysis of N -linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) by reversed-phase ultra-performance liquid chromatography with fluorescence detection and electrospray ionization time-of-flight mass spectrometry

BIOMEDICAL CHROMATOGRAPHY, Issue 5 2009
Takamasa Kurihara
Abstract Rapid, selective and sensitive determination of N -linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) was performed by ultra-performance liquid chromatography (UPLC) with fluorescence (FL) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The asparaginyl-oligosaccharide moiety was first liberated from each glycoprotein by pronase E (a proteolitic enzyme). The oligosaccharide fractions separated by gel-permeation chromatography were labeled with 1-pyrenesulfonyl chloride (PSC, a fluorescence reagent), separated by UPLC in a short run time, and then detected by FL and TOF-MS. The PSC-labeled oligosaccharides were selectively identified from the FL detection and then sensitively determined by ESI-TOF-MS. As the results, 15, eight and four kinds of N -linked oligosaccharides were detected from ovalbumin, ribonuclease B and fetuin, respectively. Because the present method is rapid (within 9 min), selective and sensitive (approximate 60 fmol, S/N = 5), the determination of N -linked oligosaccharides in various glycoproteins seems to be possible. Copyright © 2008 John Wiley & Sons, Ltd. [source]