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71.
Jennifer Dumont Don Euwart Baisong Mei Scott Estes Rashmi Kshirsagar 《Critical reviews in biotechnology》2016,36(6):1110-1122
Biotherapeutic proteins represent a mainstay of treatment for a multitude of conditions, for example, autoimmune disorders, hematologic disorders, hormonal dysregulation, cancers, infectious diseases and genetic disorders. The technologies behind their production have changed substantially since biotherapeutic proteins were first approved in the 1980s. Although most biotherapeutic proteins developed to date have been produced using the mammalian Chinese hamster ovary and murine myeloma (NS0, Sp2/0) cell lines, there has been a recent shift toward the use of human cell lines. One of the most important advantages of using human cell lines for protein production is the greater likelihood that the resulting recombinant protein will bear post-translational modifications (PTMs) that are consistent with those seen on endogenous human proteins. Although other mammalian cell lines can produce PTMs similar to human cells, they also produce non-human PTMs, such as galactose-α1,3-galactose and N-glycolylneuraminic acid, which are potentially immunogenic. In addition, human cell lines are grown easily in a serum-free suspension culture, reproduce rapidly and have efficient protein production. A possible disadvantage of using human cell lines is the potential for human-specific viral contamination, although this risk can be mitigated with multiple viral inactivation or clearance steps. In addition, while human cell lines are currently widely used for biopharmaceutical research, vaccine production and production of some licensed protein therapeutics, there is a relative paucity of clinical experience with human cell lines because they have only recently begun to be used for the manufacture of proteins (compared with other types of cell lines). With additional research investment, human cell lines may be further optimized for routine commercial production of a broader range of biotherapeutic proteins. 相似文献
72.
73.
Preparation and polymerization properties of monomeric ADP-actin 总被引:2,自引:0,他引:2
An improved method for the preparation of Mg-ADP-actin and Ca-ADP-actin which minimizes denaturation of the protein has been developed. Using ADP-actin prepared by this method, we have measured the polymerization characteristics of Mg-ADP-actin and Ca-ADP-actin. In contrast to the significant difference in Mg-ATP-actin and Ca-ATP-actin polymerization characteristics that we reported previously (J. Muscle Res. Cell Motility 7 (1986) 215-224), we show here that values for the critical concentration, the relative rate constant of elongation (mk+) and the relative rate constant of depolymerization (mk-) for Mg-ADP-actin are similar to those for Ca-ADP-actin. The value of mk+ for Mg-ATP-actin is about 8-fold higher than that for Mg-ADP-actin and the value of mk- for Mg-ADP-actin is 3-4-fold higher than that for Mg-ATP-actin. These factors may help explain the observation that the spontaneous nucleation rates of both types of ADP-actin are low in contrast to the rapid nucleation of Mg-ATP-actin. 相似文献
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77.
A study of bacterial surface oligosaccharides were investigated among
different strains of Neisseria gonorrhoeae to correlate structural features
essential for binding to the MAb 2C7. This epitope is widely expressed and
conserved in gonococcal isolates, characteristics essential to an effective
candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared
by a modification of the hot phenol-water method from which de-O-acetylated
LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and
ES-MSnin a triple quadrupole and an ion trap mass spectrometer,
respectively. Previously documented natural heterogeneity was apparent from
both LOS and OS preparations which was admixed with fragments induced by
hydrazine and mild acid treatment. Natural heterogeneity was limited to
phosphorylation and antenni extensions to the alpha-chain. Mild acid
hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic
linkage of lipid A. OS structures were determined by collisional and
resonance excitation combined with MS and multistep MSn which provided
sequence information from both neutral loss, and nonreducing terminal
fragments. A comparison of OS structures, with earlier knowledge of MAb
binding, enzyme treatment, and partial acid hydrolysis indicates a generic
overlapping domain for 2C7 binding. Reoccurring structural features include
a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the
nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc
(gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the
central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain),
moiety is required although extensions to this residue appear unnecessary.
相似文献
78.
Hanni KD Mazet JA Gulland FM Estes J Staedler M Murray MJ Miller M Jessup DA 《Journal of wildlife diseases》2003,39(4):837-850
The southern sea otter (Enhydra lutris nereis) population in California (USA) and the Alaskan sea otter (E. lutris kenyoni) population in the Aleutian Islands (USA) chain have recently declined. In order to evaluate disease as a contributing factor to the declines, health assessments of these two sea otter populations were conducted by evaluating hematologic and/or serum biochemical values and exposure to six marine and terrestrial pathogens using blood collected during ongoing studies from 1995 through 2000. Samples from 72 free-ranging Alaskan, 78 free-ranging southern, and (for pathogen exposure only) 41 debilitated southern sea otters in rehabilitation facilities were evaluated and compared to investigate regional differences. Serum chemistry and hematology values did not indicate a specific disease process as a cause for the declines. Statistically significant differences were found between free-ranging adult southern and Alaskan population mean serum levels of creatinine kinase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, calcium, cholesterol, creatinine, glucose, phosphorous, total bilirubin, blood urea nitrogen, and sodium. These were likely due to varying parasite loads, contaminant exposures, and physiologic or nutrition statuses. No free-ranging sea otters had signs of disease at capture, and prevalences of exposure to calicivirus, Brucella spp., and Leptospira spp. were low. The high prevalence (35%) of antibodies to Toxoplasma gondii in free-ranging southern sea otters, lack of antibodies to this parasite in Alaskan sea otters, and the pathogen's propensity to cause mortality in southern sea otters suggests that this parasite may be important to sea otter population dynamics in California but not in Alaska. The evidence for exposure to pathogens of public health importance (e.g., Leptospira spp., T. gondii) in the southern sea otter population, and the na?veté of both populations to other pathogens (e.g., morbillivirus and Coccidiodes immitis) may have important implications for their management and recovery. 相似文献
79.
R Graf R Mattera J Codina M K Estes L Birnbaumer 《The Journal of biological chemistry》1992,267(34):24307-24314
The baculovirus-based expression system was adapted to express alpha subunits of the complete (alpha i3) and an amino-terminally truncated (alpha i3') form of Gi3 and of two complete forms of Gs (alpha s-L and alpha s-S). Subunits encoded in full length cDNAs were obtained with yields of 40-60 mg of recombinant protein/liter of cells, of which alpha i3 was between 30 and 50% soluble, but alpha s subunits were only 5-10% soluble. Only the complete alpha i3 was myristoylated. alpha i3 was purified in four steps. The purified protein bound 0.8-0.9 mol of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) per mol of protein and had one predominant contaminant which was identified as a truncated form that begins with methionine 18 instead of methionine 1. Both the full length alpha i3 and the truncated alpha i3' formed trimers with human erythrocyte beta gamma as seen by their migration in sucrose density gradients and by an increased rate of ADP ribosylation by pertussis toxin, but compared to alpha i3, alpha i3' interacted with beta gamma with a reduced affinity and dissociated upon warming. At 32 degrees C, only full length alpha i3 was ADP-ribosylated; at 4 degrees C, alpha i3 and alpha i3' were both ADP-ribosylated, with the truncated form requiring approximately 200-fold higher concentrations of beta gamma. A genetically engineered alpha i3' (alpha i3[18-354]) was also expressed in Sf9 cells. Yields, assessed as saturable GTP gamma S binding sites, were 3-5 mg per liter. Scatchard analysis showed that truncation of the amino terminus interferes with the ability of Mg2+ to promote high affinity binding of GTP gamma S. We conclude that the G protein alpha subunit amino terminus is not essential for interaction with beta gamma dimers, but rather is important in determining the affinity of the alpha subunit for both the beta gamma dimers and guanine nucleotide. 相似文献
80.
Zongli Li Matthew L. Baker Wen Jiang Mary K. Estes B. V. Venkataram Prasad 《Journal of virology》2009,83(4):1754-1766
Rotavirus, a nonturreted member of the Reoviridae, is the causative agent of severe infantile diarrhea. The double-stranded RNA genome encodes six structural proteins that make up the triple-layer particle. X-ray crystallography has elucidated the structure of one of these capsid proteins, VP6, and two domains from VP4, the spike protein. Complementing this work, electron cryomicroscopy (cryoEM) has provided relatively low-resolution structures for the triple-layer capsid in several biochemical states. However, a complete, high-resolution structural model of rotavirus remains unresolved. Combining new structural analysis techniques with the subnanometer-resolution cryoEM structure of rotavirus, we now provide a more detailed structural model for the major capsid proteins and their interactions within the triple-layer particle. Through a series of intersubunit interactions, the spike protein (VP4) adopts a dimeric appearance above the capsid surface, while forming a trimeric base anchored inside one of the three types of aqueous channels between VP7 and VP6 capsid layers. While the trimeric base suggests the presence of three VP4 molecules in one spike, only hints of the third molecule are observed above the capsid surface. Beyond their interactions with VP4, the interactions between VP6 and VP7 subunits could also be readily identified. In the innermost T=1 layer composed of VP2, visualization of the secondary structure elements allowed us to identify the polypeptide fold for VP2 and examine the complex network of interactions between this layer and the T=13 VP6 layer. This integrated structural approach has resulted in a relatively high-resolution structural model for the complete, infectious structure of rotavirus, as well as revealing the subtle nuances required for maintaining interactions in such a large macromolecular assembly. 相似文献