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排序方式: 共有111条查询结果,搜索用时 3 毫秒
1.
Grazyna Formicka-Kozlowska Leslie D. Pettit Ian Steel Bianka Hartrodt Klaus Neubert Piotr Rekowski Gotfryd Kupryszewski 《Journal of inorganic biochemistry》1984,22(3):155-163
The synthesis of β-casomorphin-5 (Tyr-Pro-Phe-Pro-Gly, H2L) and a number of its peptide fragments is described. Complexes formed between these peptides and Cu(II) have been investigated spectrophotometrically, using CD and EPR spectroscopy, and potentiometrically. Results show that, with tyrosine as the N-terminal residue, the major complex formed at physiological pH is the dimeric species, [Cu2L2], bonded through the phenolic O? of the Tyr residue of one ligand and the N-terminal amine nitrogen of the second ligand molecule. There is no evidence for coordination through the peptide nitrogens unless the terminal Tyr group is removed. 相似文献
2.
The yeast lgl family member Sro7p is an effector of the secretory Rab GTPase Sec4p 总被引:1,自引:0,他引:1
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Grosshans BL Andreeva A Gangar A Niessen S Yates JR Brennwald P Novick P 《The Journal of cell biology》2006,175(1):55-66
Rab guanosine triphosphatases regulate intracellular membrane traffic by binding specific effector proteins. The yeast Rab Sec4p plays multiple roles in the polarized transport of post-Golgi vesicles to, and their subsequent fusion with, the plasma membrane, suggesting the involvement of several effectors. Yet, only one Sec4p effector has been documented to date: the exocyst protein Sec15p. The exocyst is an octameric protein complex required for tethering secretory vesicles, which is a prerequisite for membrane fusion. In this study, we describe the identification of a second Sec4p effector, Sro7p, which is a member of the lethal giant larvae tumor suppressor family. Sec4-GTP binds to Sro7p in cell extracts as well as to purified Sro7p, and the two proteins can be coimmunoprecipitated. Furthermore, we demonstrate the formation of a ternary complex of Sec4-GTP, Sro7p, and the t-SNARE Sec9p. Genetic data support our conclusion that Sro7p functions downstream of Sec4p and further imply that Sro7p and the exocyst share partially overlapping functions, possibly in SNARE regulation. 相似文献
3.
Hussam H. Shaheen Bianka Prinz Ming-Tang Chen Tej Pavoor Song Lin Nga Rewa Houston-Cummings Renee Moore Terrance A. Stadheim Dongxing Zha 《PloS one》2013,8(7)
State-of-the-art monoclonal antibody (mAb) discovery methods that utilize surface display techniques in prokaryotic and eukaryotic cells require multiple steps of reformatting and switching of hosts to transition from display to expression. This results in a separation between antibody affinity maturation and full-length mAb production platforms. Here, we report for the first time, a method in Glyco-engineered
Pichia
pastoris
that enables simultaneous surface display and secretion of full-length mAb molecules with human-like N-glycans using the same yeast cell. This paradigm takes advantage of homo-dimerization of the Fc portion of an IgG molecule to a surface-anchored "bait" Fc, which results in targeting functional “half” IgGs to the cell wall of
Pichia
pastoris
without interfering with the secretion of full length mAb. We show the utility of this method in isolating high affinity, well-expressed anti-PCSK9 leads from a designed library that was created by mating yeasts containing either light chain or heavy chain IgG libraries. Coupled with Glyco-engineered
Pichia
pastoris
, this method provides a powerful tool for the discovery and production of therapeutic human mAbs in the same host thus improving drug developability and potentially shortening the discovery time cycle. 相似文献
4.
Prinz B Schultchen J Rydzewski R Holz C Boettner M Stahl U Lang C 《Journal of structural and functional genomics》2004,5(1-2):29-44
We describe the introduction of the yeasts Saccharomyces cerevisiae and Pichia pastoris as eukaryotic hosts for the routine production of recombinant proteins for a structural genomics initiative. We have previously shown that human cDNAs can be efficiently expressed in both hosts using high throughput procedures. Expression clones derived from these screening procedures were grown in bioreactors and the over-expressed human proteins were purified, resulting in obtaining significant amounts suitable for structural analysis. We have also developed and optimized protocols enabling a high throughput, low cost fermentation and purification strategy for recombinant proteins for both S. cerevisiae and P. pastoris on a scale of 5 to 10 mg. Both batch and fed batch fermentation methods were applied to S. cerevisiae. The fed batch fermentations yielded a higher biomass production in all the strains as well as a higher productivity for some of the proteins. We carried out only fed batch fermentations on P. pastoris strains. Biomass was produced by cultivation on glycerol, followed by feeding methanol as carbon source to induce protein expression. The recombinant proteins were expressed as fusion proteins that include a N-terminal His-tag and a C-terminal Strep-tag. They were then purified by a two-step chromatographic procedure using metal-affinity chromatography and StrepTactin-affinity chromatography. This was followed by gel filtration for further purification and for buffer exchange. This three-step purification procedure is necessary to obtain highly purified proteins from yeast. The purified proteins have successfully been subjected to crystallization and biophysical analysis. 相似文献
5.
6.
Bianka Mußil Ulrike Sauermann Dirk Motzkus Christiane Stahl-Hennig Sieghart Sopper 《Retrovirology》2011,8(1):1-17
Background
The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM).Results
Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6.Conclusions
In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential. 相似文献7.
Jankowska Urszula Skupien-Rabian Bozena Swiderska Bianka Prus Gabriela Dziedzicka-Wasylewska Marta Kedracka-Krok Sylwia 《Neurochemical research》2021,46(8):2097-2111
Neurochemical Research - Although antipsychotics are routinely used in the treatment of schizophrenia for the last decades, their precise mechanism of action is still unclear. In this study, we... 相似文献
8.
Epidermal cell death in rice is regulated by ethylene, gibberellin, and abscisic acid 总被引:8,自引:0,他引:8
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Programmed cell death (PCD) of epidermal cells that cover adventitious root primordia in deepwater rice (Oryza sativa) is induced by submergence. Early suicide of epidermal cells may prevent injury to the growing root that emerges under flooding conditions. Induction of PCD is dependent on ethylene signaling and is further promoted by gibberellin (GA). Ethylene and GA act in a synergistic manner, indicating converging signaling pathways. Treatment of plants with GA alone did not promote PCD. Treatment with the GA biosynthesis inhibitor paclobutrazol resulted in increased PCD in response to ethylene and GA presumably due to an increased sensitivity of epidermal cells to GA. Abscisic acid (ABA) was shown to efficiently delay ethylene-induced as well as GA-promoted cell death. The results point to ethylene signaling as a target of ABA inhibition of PCD. Accumulation of ethylene and GA and a decreased ABA level in the rice internode thus favor induction of epidermal cell death and ensure that PCD is initiated as an early response that precedes adventitious root growth. 相似文献
9.
Tollinger M Kloiber K Agoston B Dorigoni C Lichtenecker R Schmid W Konrat R 《Biochemistry》2006,45(29):8885-8893
NMR relaxation dispersion techniques were used to investigate conformational exchange of the three-helix bundle protein KIX under native conditions. These experiments provide site-resolved kinetic information about microsecond-to-millisecond time scale motions along with structural (chemical shift) information without requiring a perturbation of the equilibrium. All kinetic data are consistent with an apparent two-state transition between natively folded KIX and a partially unfolded high-energy state that is populated to 3.0 +/- 0.2% at 27 degrees C. By combining (13)C- and (15)N-based experiments that probe specific structural aspects, we show that the sparsely populated high-energy state displays a strong conformational preference. An isolated secondary structural element, C-terminal helix alpha3, is highly populated, while the hydrophobic core of the domain and the remainder of the protein backbone, including helices alpha1 and alpha2, are disordered and devoid of specific interactions. This high-energy state presumably represents the equilibrium analogue of a folding intermediate that is transiently populated in stopped-flow kinetic experiments [Horng, J. C., Tracz, S. M., Lumb, K. J., and Raleigh, D. P. (2002) Biochemistry 44, 627-634]. 相似文献
10.
Li H Sethuraman N Stadheim TA Zha D Prinz B Ballew N Bobrowicz P Choi BK Cook WJ Cukan M Houston-Cummings NR Davidson R Gong B Hamilton SR Hoopes JP Jiang Y Kim N Mansfield R Nett JH Rios S Strawbridge R Wildt S Gerngross TU 《Nature biotechnology》2006,24(2):210-215
As the fastest growing class of therapeutic proteins, monoclonal antibodies (mAbs) represent a major potential drug class. Human antibodies are glycosylated in their native state and all clinically approved mAbs are produced by mammalian cell lines, which secrete mAbs with glycosylation structures that are similar, but not identical, to their human counterparts. Glycosylation of mAbs influences their interaction with immune effector cells that kill antibody-targeted cells. Here we demonstrate that human antibodies with specific human N-glycan structures can be produced in glycoengineered lines of the yeast Pichia pastoris and that antibody-mediated effector functions can be optimized by generating specific glycoforms. Glycoengineered P. pastoris provides a general platform for producing recombinant antibodies with human N-glycosylation. 相似文献