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1.
Yoshikazu Hasegawa Sumio Nakamura Sayuri Kakizoe Masayuki Sato Norio Nakamura 《Journal of plant research》1998,111(3):421-429
As a first step towards studying the biochemical relationship between Golgi vesicles (GVs) and tube wall components, isolation
of GVs from the growing pollen tubes ofCamellia japonica was attempted using a centrifugation method with mannitol. The isolated GV was identified ultrastructurally and immunocytochemically.
The main components of the GV were proteins and carbohydrates. The main monosaccharides of GV polysaccharides were galactose,
arabinose and uronic acid, and pectins and arabinogalactan proteins also were detected immunochemically. An antiserum against
the isolated GVs reacted with the outer layer of the pollen tube wall and the intine layers of the grain wall as well as thein situ GVs in the pollen tube and the grain cytoplasm. We have thus successfully isolated GVs and shown that they contain pectic
substances and arabinogalactan proteins which contribute to formation of the pollen tube primary wall. 相似文献
2.
Li Meng Jean Rutledge Ying Zhu Gerald M. Kidder Firouz Khamsi David T. Armstrong 《Molecular reproduction and development》1996,43(2):228-235
To investigate the role of the germinal vesicle (GV) on in vitro maturation (IVM) of rat oocytes, we examined protein synthesis during IVM by comparing polypeptide patterns in control and enucleated oocytes using one and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separation of polypeptides extracted from the cytoplasm of GV by one-dimensional SDS-PAGE revealed that a 55 kDa polypeptide was present only in the GVs of rat oocytes. At 0, 12, 24, 36, and 44 hr after PMSG injection, prior to the initiation of maturation, enucleated oocytes synthesized the same major polypeptides as cumulus intact (CI) oocytes. During meiotic maturation, no major changes were detected in protein synthesis from prophase (GV stage) to prometaphase I (0–6 hr IVM). However, after entry into prometaphase I (7 hr IVM), striking changes were seen; a 24 kDa polypeptide disappeared and expression of a 34 kDa polypeptide became stronger. This pattern lasted until metaphase II. We detected no major differences in the pattern of protein synthesis between CI and enucleated oocytes using two-dimensional PAGE. These results indicate that protein synthesis in the maturing rat oocyte is controlled by cytoplasmic regulators rather than intrinsic nuclear components. © 1996 Wiley-Liss, Inc. 相似文献
3.
Erin Quartley Andrei Alexandrov Maryann Mikucki Frederick S. Buckner Wim G. Hol George T. DeTitta Eric M. Phizicky Elizabeth J. Grayhack 《Journal of structural and functional genomics》2009,10(3):233-247
High level expression of many eukaryotic proteins for structural analysis is likely to require a eukaryotic host since many
proteins are either insoluble or lack essential post-translational modifications when expressed in E. coli. The well-studied eukaryote Saccharomyces cerevisiae possesses several attributes of a good expression host: it is simple and inexpensive to culture, has proven genetic tractability,
and has excellent recombinant DNA tools. We demonstrate here that this yeast exhibits three additional characteristics that
are desirable in a eukaryotic expression host. First, expression in yeast significantly improves the solubility of proteins
that are expressed but insoluble in E. coli. The expression and solubility of 83 Leishmania major ORFs were compared in S. cerevisiae and in E. coli, with the result that 42 of the 64 ORFs with good expression and poor solubility in E. coli are highly soluble in S. cerevisiae. Second, the yield and purity of heterologous proteins expressed in yeast is sufficient for structural analysis, as demonstrated
with both small scale purifications of 21 highly expressed proteins and large scale purifications of 2 proteins, which yield
highly homogeneous preparations. Third, protein expression can be improved by altering codon usage, based on the observation
that a codon-optimized construct of one ORF yields three-fold more protein. Thus, these results provide direct verification
that high level expression and purification of heterologous proteins in S. cerevisiae is feasible and likely to improve expression of proteins whose solubility in E. coli is poor. 相似文献
4.
Heterologous protein production in yeast 总被引:5,自引:0,他引:5
Gerd Gellissen Karl Melber Zbigniew A. Janowicz Ulrike M. Dahlems Ulrike Weydemann Michael Piontek Alexander W. M. Strasser Cornelis P. Hollenberg 《Antonie van Leeuwenhoek》1992,62(1-2):79-93
The exploitation of recombinant DNA technology to engineer expression systems for heterologous proteins represented a major task within the field of biotechnology during the last decade. Yeasts attracted the attention of molecular biologists because of properties most favourable for their use as hosts in heterologous protein production. Yeasts follow the general eukaryotic posttranslational modification pattern of expressed polypeptides, exhibit the ability to secrete heterologous proteins and benefit from an established fermentation technology. Aside from the baker's yeastSaccharomyces cerevisiae, an increasing number of alternative non-Saccharomyces yeast species are used as expression systems in basic research and for an industrial application.In the following review a selection from the different yeast systems is described and compared. 相似文献
5.
The methylotrophic yeast Pichia pastoris is a popular heterologous expression host for the recombinant production of a variety of prokaryotic and eukaryotic proteins.
The rapid emergence of P. pastoris as a robust heterologous expression host was facilitated by the ease with which it can be manipulated and propagated, which
is comparable to that of Escherichia coli and Saccharomyces cerevisiae. P. pastoris offers further advantages such as the tightly-regulated alcohol oxidase promoter that is particularly suitable for heterologous
expression of foreign genes. While recombinant production of bacterial toxins and their derivatives is highly desirable, attempts
at their heterologous expression using the traditional E. coli expression system can be problematic due to the formation of inclusion bodies that often severely limit the final yields
of biologically active products. However, recent literature now suggests that P. pastoris may be an attractive alternative host for the heterologous production of bacterial toxins, such as those from the genera
Bacillus, Clostridium, and Corynebacterium, as well as their more complex derivatives. Here, we review the recombinant production of bacterial toxins and their derivatives
in P. pastoris with special emphasis on their potential clinical applications. Considering that de novo design and construction of synthetic toxin genes have often been necessary to achieve optimal heterologous expression in
P. pastoris, we also present general guidelines to this end based on our experience with the P. pastoris expression of the Bacillus thuringiensis Cyt2Aa1 toxin. 相似文献
6.
米曲霉作为一种重要的工业微生物,在异源蛋白表达方面已有广泛应用,受限于被表达蛋白的修饰及分泌过程,目前实际生产使用的基因供体主要局限于其他真菌,尤其是丝状真菌。当外源基因来源于植物、昆虫和哺乳动物时,米曲霉所生产的异源蛋白产量及生物活性往往不尽如人意。本文综述了米曲霉作为宿主表达异源蛋白的研究进展,包括其现有的遗传操作手段及异源表达方面的应用及探索,重点介绍了应用过程中面临的挑战和解决策略,另外,对米曲霉表达异源蛋白的应用前景及发展方向进行了展望。 相似文献
7.
生发泡(germinal vesicle,GV)移植到去核的GV期卵母细胞后,获得重构卵,重构卵在体外能成熟,受精和进行胚胎发育。GV移植到去核的第二次减数分裂中期(metaphase Ⅱ,MII)卵母细胞后,重构卵能发生GV破裂,但难以排出第一极体。GV移植后,通过连续核移植,重构合子具有发育到终期的能力。GV移植为研究卵母细胞的发育提供了一种重要工具。 相似文献
8.
The food-grade status and probiotic activity of lactic acid bacteria (LAB) make them attractive hosts for production and oral delivery of therapeutic heterologous vaccines and other proteins, yet these bacteria currently do not achieve recombinant protein expression at levels comparable to those seen in Escherichia coli and Saccharomyces cerevisiae. Limited levels of expressed recombinant protein per cell most likely constrain the vaccine’s immunogenic potential with respect to the magnitude and specificity of the immune response. With the goal of increasing recombinant protein expression per cell in Lactococcus lactis IL1403, a model LAB, we have constructed and evaluated a new vector that permits simultaneously-induced expression of GFP, a model recombinant protein, and antisense RNA inhibition of the clpP-encoded intracellular protease. While silencing of the rational target clpP does not lead to increased GFP per cell, the new dual-expression system provides an efficient and potentially high-throughput metabolic engineering tool for strain improvement. 相似文献
9.
Saccharomyces cerevisiae is frequently used in biotechnology, including fermentative processes in food production, heterologous protein production
and high throughput developments for biomedicine. Accurate expression of selected genes is essential for all these areas.
Systems that can be regulated are particularly useful because they allow controlling the timing and levels of gene expression.
We examine here new expression systems that have been described, including improvements of classical ones and new strategies
of artificial gene control that have been applied in functional genomics. 相似文献
10.
《Journal of receptor and signal transduction research》2013,33(1):61-73
AbstractWe have produced a plasmid designed for the expression of heterologous G protein α subunits in the yeast Saccharomyces cerevisiae Introduction of these genes is by simple cassette replacement using unique restriction sites, and their expression is controlled by the regulatory sequences of the S. cerevisiae GPA1 gene. Levels of expression are therefore suitable for interaction of these heterologous proteins with elements of the yeast pheromone response pathway. We believe that this plasmid will facilitate the coupling of more members of the seven transmembrane domain superfamily of receptors, through their native G protein α subunit, to the yeast pheromone response pathway.The plasmid pRGP, is a stable centromeric shuttle vector with a HIS3-selectable marker. We have demonstrated that production of GPA1 from this plasmid functionally complements a gpal- null mutation. A similar response is obtained when an alternative G protein a subunit, Golf, is introduced using pRGP. We believe that this is the first example of a heterologous G protein shown to couple to a yeast pheromone receptor. 相似文献
11.
Display of heterologous proteins on the Saccharomyces cerevisiae surface display system using a single constitutive expression vector 下载免费PDF全文
Jie Zhang Qing Liu Longjiang Wang Peipei Chen Fangkun Wang Hongmei Li Yihong Xiao Xiaomin Zhao 《Biotechnology progress》2014,30(2):443-450
In this study, we constructed a novel and simple yeast surface display system with a single expression vector. The newly established system uses a bidirectional expression vector carrying the AGA1 gene driven by the PGK1 promoter in one direction and the AGA2‐expression cassette driven by the TEF1 promoter in the reverse direction, and uses the geneticin, a G418‐resistant gene, as the selection marker for transformants. Because all the display elements are put into one expression vector, the new system is much simpler to use, and there is no need for any genetic modification of the host strains; therefore, the new system can be used in wild type as well as laboratory strains of Saccharomyces cerevisiae. The display efficiency of heterologous proteins using the new system has been confirmed by displaying enhanced green fluorescent protein and Eimeria tenella (a chicken protozoan parasite) microneme protein2 (EtMic2) on several S. cerevisiae strains. We also tested the new system with an aga2 mutant strain of S. cerevisiae. The results indicate that the native expressed Aga2 protein has no effect on the display efficiency of heterologous proteins. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:443–450, 2014 相似文献
12.
W. F. Li J. Ji G. Wang H. Y. Wang B. L. Niu T. L. Josine 《Russian Journal of Genetics》2011,47(9):1039-1046
Many natural proteins have been developed into drugs and produced for direct application. Identifying improved hosts to achieve
high-level heterologous protein production is a challenge in the study of heterologous protein expression in recombinant yeast.
In this study, a novel high-throughput assay to screen such overproducing Saccharomyces cerevisiae strains was systematically developed. The protocol designed was based on screening host strain derivatives with increased
superoxide dismutase dependent resistance to oxidative stress. Yeast cells transformed with recombinant plasmid carrying SOD1 gene as a reporter responded exquisitely to oxidative stress induced by elevated concentrations of paraquat. Improved yeast
strains resulting from screening clones subjected to genome shuffling through selective pressure argue for a more effective
screening system compared with traditonal selection. Moreover, this approach can be employed in general biochemical analysis
without utilization of flow cytometry or well plate reader. Therefore, it is expected that the high-throughput assay would
make superior strains producing heterologous proteins. 相似文献
13.
气囊(gas vesicles,GVs)是一种存在于蓝藻及古菌等微生物中调节浮力的类细胞器纳米结构,由蛋白质外壳包裹气体组成。近年来的研究表明,气囊具有作为超声分子影像探针的潜力。然而,气囊的充放气机制并不明确,限制了生物合成超声分子影像探针的保存和气体更换。本研究发现环境pH值是调节气囊充放气的一个重要因素。其不仅可以调节藻细胞内的气囊充放气进而使微囊藻呈现不同的漂浮状态,还可对提纯的气囊充放气进行体外调节,且该调节过程可逆。该机制的阐明为生物合成超声分子影像探针的大规模生产和保存,特别对气囊中的气体进行更换以满足不同的诊疗需求提供了技术支持,助力生物合成超声造影剂在疾病诊疗中的应用。 相似文献
14.
We have analyzed the intracellular behavior of the human transferrin receptor (TfR) in Saccharomyces cerevisiae. The major part of the heterologously expressed TfR, which has previously been used as a model for heterologous expression
of membrane proteins in yeast, is localized in the endoplasmic reticulum (ER) membranes; a minor fraction is present in the
plasma membrane (PM). The stability of the TfR depends on vacuolar proteases, implying that it is degraded in the vacuolar
compartment. Degradation is further dependent on favorable transport conditions to this compartment. The main bottleneck of
transport seems to be the transition from the ER to the PM. The chaperone Cne1p, which is involved in quality control in the
ER, plays a role in regulating the amount of heterologous TfR, as deletion of CNE1 leads to significant accumulation of the protein. This is the first demonstration of the involvement of CNE1 in regulating the level of heterologous membrane proteins.
Electronic Publication 相似文献
15.
16.
Irfan Farabi Hayat Manuel Plan Birgitta E. Ebert Geoff Dumsday Claudia E. Vickers Bingyin Peng 《Microbial biotechnology》2021,14(6):2627-2642
The yeast Saccharomyces cerevisiae uses the pyruvate dehydrogenase-bypass for acetyl-CoA biosynthesis. This relatively inefficient pathway limits production potential for acetyl-CoA-derived biochemical due to carbon loss and the cost of two high-energy phosphate bonds per molecule of acetyl-CoA. Here, we attempted to improve acetyl-CoA production efficiency by introducing heterologous acetylating aldehyde dehydrogenase and phosphoketolase pathways for acetyl-CoA synthesis to enhance production of the sesquiterpene trans-nerolidol. In addition, we introduced auxin-mediated degradation of the glucose-dependent repressor Mig1p to allow induced expression of GAL promoters on glucose so that production potential on glucose could be examined. The novel genes that we used to reconstruct the heterologous acetyl-CoA pathways did not sufficiently complement the loss of endogenous acetyl-CoA pathways, indicating that superior heterologous enzymes are necessary to establish fully functional synthetic acetyl-CoA pathways and properly explore their potential for nerolidol synthesis. Notwithstanding this, nerolidol production was improved twofold to a titre of ˜ 900 mg l−1 in flask cultivation using a combination of heterologous acetyl-CoA pathways and Mig1p degradation. Conditional Mig1p depletion is presented as a valuable strategy to improve the productivities in the strains engineered with GAL promoters-controlled pathways when growing on glucose. 相似文献
17.
《Bioscience, biotechnology, and biochemistry》2013,77(3):628-631
In this study, the production of eight G protein-coupled receptors by Saccharomyces cerevisiae was compared using two types of media, one of which contained soy peptides and the other free amino acids. Yeast cell growth improved in the medium with soy peptides, and the expression levels of six of the receptors increased during the exponential phase by an average of 2.3-fold as against the free amino acid-based medium. The enhancement of protein expression by soy peptides can be explained by alleviation of metabolite stress due to amino acid source depletion caused by heterologous protein expression. 相似文献
18.
Xiuxia Liu Wei Zhang Zihao Zhao Xiaofeng Dai Yankun Yang 《Critical reviews in biotechnology》2017,37(4):541-551
Corynebacterium glutamicum, a Gram-positive bacterium, has been widely used for the industrial production of amino acids, such as glutamate and lysine, for decades. Due to several characteristics – its ability to secrete properly folded and functional target proteins into culture broth, its low levels of endogenous extracellular proteins and its lack of detectable extracellular hydrolytic enzyme activity – C. glutamicum is also a very favorable host cell for the secretory production of heterologous proteins, important enzymes, and pharmaceutical proteins. The target proteins are secreted into the culture medium, which has attractive advantages over the manufacturing process for inclusion of body expression – the simplified downstream purification process. The secretory process of proteins is complicated and energy consuming. There are two major secretory pathways in C. glutamicum, the Sec pathway and the Tat pathway, both have specific signal peptides that mediate the secretion of the target proteins. In the present review, we critically discuss recent progress in the secretory production of heterologous proteins and examine in depth the mechanisms of the protein translocation process in C. glutamicum. Some successful case studies of actual applications of this secretory expression host are also evaluated. Finally, the existing issues and solutions in using C. glutamicum as a host of secretory proteins are specifically addressed. 相似文献
19.
Grard Coffe Marie-Noëlle Raymond 《Biology of the cell / under the auspices of the European Cell Biology Organization》1990,70(3):143-152
Summary— We report an isolation procedure of trans-Golgi vesicles (GVs) from rat parotid glands. Various organelle markers were used, particularly galactosyl transferase as a trans-Golgi marker, to test the purity of the GV fraction. A quantitative in vitro binding assay between microtubules and GVs is described. The vesicles were incubated with taxol-induced microtubules, layered between 50% and 43% sucrose cushions and subjected to centrifugation. Unlike free microtubules which were sedimented, the GV-bound microtubules co-migrated upward with GVs. Quantification of these bound microtubules was carried out by densitometric scanning of Coomassie blue-stained gels. The association between microtubules and GVs followed a saturation curve, with a plateau value of 20 μg of microtubule protein bound to 500 μg of GV fraction. The half-saturation of the GV sites was obtained with a microtubule concentration of 20 μg/ml. Electron microscopy of negatively stained re-floated material showed numerous microtubule-vesicle complexes. Coating of microtubules with an excess of brain microtubule-associated proteins (MAPs) abolished binding. In the absence of exogenous microtubules, we showed that the GV fraction was already interacting with a class of endogenous rat parotid microtubules. This class of colcemid and cold-stable microtubules represents 10–20% of the total tubulin content of the parotid cell. 相似文献