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Haojie Yu Xin Yan Weiliang Shen Qing Hong Ji Zhang Yujia Shen Shunpeng Li 《Current microbiology》2009,59(6):573-578
In the Pichia pastoris expression system, increasing the copy number of the expression cassette often has the effect of increasing the amount of
protein expressed. To improve the expression level of methyl parathion hydrolase (MPH), we constructed two integration vectors
with four and eight direct repeats of the expression cassette using an in vitro multimerization approach. After two successive
integrations, at least 12 copies of the MPH expression cassette were integrated into the P. pastoris chromosome. Under shake-flask conditions, over 55 mg active MPH/l was secreted into the medium by the multicopy clones. The
extracellular enzyme activity was about 10-fold higher for the multicopy clones than for clones containing a single copy of
the gene. Further investigations revealed that the multicopy MPH expression cassette could remain stably integrated and functional
over five generations. Note that the expression vector pRF constructed in our study can be not only used to construct multiple
copies of the expression cassette in vitro, but also integrated into the P. pastoris genome without introducing any antibiotic resistance gene, which is desirable for production of biotherapeutic proteins. 相似文献
3.
Tatina T. Todorova Ventsislava Y. Petrova Stéphane Vuilleumier Anna V. Kujumdzieva 《Archives of microbiology》2009,191(11):837-845
Growth of Saccharomyces cerevisiae
ure2Δ mutant strain was investigated in the presence of diverse oxidant compounds. The inability of the strain to grow on a medium
supplemented with H2O2 was confirmed and a relationship between diminishing levels of glutathione (GSH) and peroxide sensitivity was established.
Data for the lack of significant effect of URE2 disruption on the cellular growth in the presence of paraquat and menadione were obtained. The possible role of Ure2p in
acquiring sensitivity to oxidative stress by means of its regulatory role in the GATA signal transduction pathway was discussed.
It was suggested that the susceptibility of ure2Δ mutant to the exogenous hydrogen peroxide can result from increased GSH degradation due to the deregulated localization of
the γ-glutamyl transpeptidase activating factors Gln3/Gat1. The important role of Ure2p in in vivo glutathione-mediated reactive
oxygen species (ROS) scavenging was shown by measuring the activity of antioxidant enzymes glutathione peroxidase, superoxide
dismutase (SOD) and catalase in an URE2 disrupted strain. A time-dependent increase in SOD and catalase activity was observed. More importantly, it was shown that
the ure2 mutation could cause significant disturbance in cellular oxidant balance and increased ROS level. 相似文献
4.
Background
High cell density cultures of Pichia pastoris grown on methanol tend to develop yellow colored supernatants, attributed to the release of free flavins. The potential of P. pastoris for flavin overproduction is therefore given, but not pronounced when the yeast is grown on glucose. The aim of this study is to characterize the relative regulatory impact of each riboflavin synthesis gene. Deeper insight into pathway control and the potential of deregulation is established by overexpression of the single genes as well as a combined deregulation of up to all six riboflavin synthesis genes. 相似文献5.
Transglutaminase-like activity participates in cell wall biogenesis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 总被引:1,自引:0,他引:1
Transglutaminases (TGases) catalyze the cross-linking between protein molecules by formation of an amide bond between γ-carboxyamide
group of glutamine and the ε-amine group of lysine under deamination of glutamine. We have demonstrated the participation
of transglutaminase-like activity in the isolated cell walls and in the process of cell wall regeneration in protoplasts of
the yeast Saccharomyces cerevisiae. A radioactive TGase substrate [3H]putrescine was incorporated into the isolated cell walls and into the TCA-insoluble fraction in regenerating protoplasts.
The incorporation was increased by adding exogenous artificial substrate of TGase N,N’-dimethylcasein and was inhibited by
TGase inhibitor cystamine and/or EDTA. These results suggest the existence of a TGase-type reaction involved in the formation
of covalent cross-links between glycoprotein molecules during cell wall construction in S. cerevisiae. 相似文献
6.
POL LHOAS 《Nature: New biology》1972,236(64):86-87
IN contrast to bacteriophages which are strictly host-specific, double stranded RNA fungal viruses are shown to be able to infect a different host genus and so bear some similarity to some viruses of higher plants. The technique of infecting yeasts with viruses from filamentous fungi and the morphological criterion by which infection is detected are described here. 相似文献
7.
I. A. Sevostyanova V. A. Selivanov V. A. Yurshev O. N. Solovjeva S. V. Zabrodskaya G. A. Kochetov 《Biochemistry. Biokhimii?a》2009,74(7):789-792
Catalytic activity of two active sites of transketolase and their affinity towards the substrates (xylulose-5-phosphate and ribose-5-phosphate) has been studied in the presence of Ca2+ and Mg2+. In the presence of Ca2+, the active sites exhibit negative cooperativity in binding both xylulose-5-phosphate (donor substrate) and ribose-5-phosphate (acceptor substrate) and positive cooperativity in the catalytic transformation of the substrates. In the presence of Mg2+, nonequivalence of the active sites is not observed. 相似文献
8.
There are three most important bacterial causative agents of serious infections that could be misused for warfare purposes:
Bacillus anthracis (the causative agent of anthrax) is the most frequently mentioned one; however, Fracisella tularensis (causing tularemia) and Yersinia pestis (the causative agent of plague) are further bacterial agents enlisted by Centers for Disease Control and Prevention into the category A of potential biological weapons. This review intends to summarize basic information about these bacterial
agents. Military aspects of their pathogenesis and the detection techniques suitable for field use are discussed. 相似文献
9.
A cell surface display system was developed in Pichia pastoris using the gene TIP1, encoding the glycosylphosphatidylinositol (GPI)-anchored protein of Saccharomyces cerevisiae (ScTIP). Human lactoferrin cDNA (hLf) was fused to a full-length TIP1 DNA (ScTIP
630
) or a short-TIP1 fragment (ScTIP
120
) encoding the 40 C-terminal amino acids of ScTIP. Both hLf-ScTIP fusion genes were expressed in P. pastoris SMD 1168. The fused protein was detected by western blotting after extraction of the lysed recombinant cells with Triton
X-100, urea, and Triton X-100 plus urea, suggesting that the hLf is associated with the membrane. The localization of surface-displayed
hLf was confirmed by immunofluorescence confocal microscopy and flow cytometric analysis using FITC-labeled anti-hLf antibody,
suggesting that hLf was successfully located at the surface of P. pastoris. The intact recombinant cells and cell lysates showed antibacterial activity against target microorganisms, meaning that
the expressed hLf was biologically active. The results indicated that the ScTIP anchoring motif is useful for cell surface
display of foreign proteins in P. pastoris. 相似文献
10.
O. A. Esakova L. E. Meshalkina G. A. Kochetov R. Golbik 《Biochemistry. Biokhimii?a》2009,74(11):1234-1238
Pyruvate derivatives halogenated at C3 were shown to be donor substrates in the transketolase reaction. No drastic differences
between the derivatives were observed in the value of the catalytic constant, whereas the Michaelis constant increased in
the following order: Br-pyruvate < Cl-pyruvate < Cl2-pyruvate < F-pyruvate < Br2-pyruvate. The presence of the halogenated pyruvate derivatives increased the affinity of apotransketolase for the coenzyme;
of note, the extent of this effect was equal with both of the active centers of the enzyme. In contrast, the presence of any
other substrate known to date, including hydroxypyruvate (i.e. pyruvate hydroxylated at C3), induced nonequivalence of the
active centers in that they differed in the extent to which the affinity for the coenzyme increased. Consequently, the β-hydroxyl
of dihydroxyethylthiamine diphosphate (an intermediate of the transketolase reaction) played an important role in the phenomenon
of non-equivalence of the active centers associated with the coenzyme binding. The fundamental possibility was demonstrated
of using halogenated pyruvate derivatives as donors of the halogen-hydroxyethyl group in organic synthesis of halogenated
carbohydrates involving transketolase. 相似文献
11.
Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo 1p with
its own secretion signal sequence or the α-factor secretion signal sequence, a polyhistidine (6×His) tag for detection, an
enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial
peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy
and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after
proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR. 相似文献
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13.
Jihee Yoon Jae-Min Park Seung-Ki Jung Keun-Young Kim Yang-Hoon Kim Jiho Min 《Current microbiology》2009,59(1):48-52
The antimicrobial activity of lysosomes, a cell organelle, against a range of test microorganisms was examined in this study.
The lysosomes isolated from Saccharomyces cerevisiae showed antimicrobial activity to Escherichia coli that positively correlated with the pH of the phosphate buffer as a dissolving solvent. The lysosomes from S. cerevisiae exhibited optimal activity at a concentration of 40%, at pH 4.0 of phosphate buffer, and at broad range temperature, except
of over 50°C. It was also found that the lysosomes have antimicrobial activity against seven different microorganisms including
E. coli. In addition, S. cerevisiae were exposed by a treatment with H2O2 and lysosomes were isolated from H2O2 exposed S. cerevisiae. We found that fluorescent intensities of each isolated lysosomes were increased depending on the increment of treated H2O2 concentration, and the lysosomes from 20 mM H2O2 treated S. cerevisiae showed higher antimicrobial activity than those from normal S. cerevisiae. Therefore, it suggests that lysosomes isolated from S. cerevisiae can be used as an antimicrobial agent. In addition, lysosomes activated by H2O2 enhanced its antimicrobial activity. 相似文献
14.
The ability of added acetaldehyde to stimulate growth in ethanol-stressed Saccharomyces cerevisiae while grown on non-fermentable substrates (ethanol, glycerol) is reported. The addition of acetaldehyde to ethanol-stressed
yeast grown on either ethanol or glycerol led to a significant decrease in lag time of 67 and 45 %, respectively (p = 0.000) and an increase in the specific growth rate (0.008–0.038/h and 0.060–0.074/h, respectively). The stimulatory effect
of acetaldehyde could be mimicked by the addition of propionaldehyde. Results, following metabolic tracing of the added stimulants,
question the previously held notion that the acetaldehyde effect in S. cerevisiae is fully redox related. 相似文献
15.
O. V. Golovanova V. I. Konenkov A. V. Shevchenko M. V. Smolnikova 《Russian Journal of Genetics》2009,45(8):981-986
Based on population analysis of the DRB1, DQA1, DQB1 and TNFA allele frequency distribution patterns, regional features of immunogenetic structure of the population of West Siberia were
investigated. Statistically significant linkage disequilibrium within the HLA class II region, as well as between the TNFA and DRB1, DQA1, and DQB1 was demonstrated. Population frequency distribution patterns of two- and multilocus haplotypes were examined. 相似文献
16.
Junmei Ding Xiaowei Huang Lemin Zhang Na Zhao Dongmei Yang Keqin Zhang 《Applied microbiology and biotechnology》2009,85(2):253-263
Eukaryotic cells have developed diverse strategies to combat the harmful effects of a variety of stress conditions. In the
model yeast Saccharomyces cerevisiae, the increased concentration of ethanol, as the primary fermentation product, will influence the membrane fluidity and be
toxic to membrane proteins, leading to cell growth inhibition and even death. Though little is known about the complex signal
network responsible for alcohol stress responses in yeast cells, several mechanisms have been reported to be associated with
this process, including changes in gene expression, in membrane composition, and increases in chaperone proteins that help
stabilize other denatured proteins. Here, we review the recent progresses in our understanding of ethanol resistance and stress
responses in yeast. 相似文献
17.
The intron sequence of chloroplast rpS16 and the secondary structure of its pre-mRNA were characterized for the first time in 26 Allium sativum accessions of different ecologo-geographical origins and seven related Allium species. The boundaries and main stem-loop consensus sequences were identified for all six domains of the intron. Polymorphism
was estimated for the total intron and its regions. The structural regions of the rpS16 intron proved to be heterogeneous for mutation rate and spectrum. Mutations were most abundant in domains II and IV, and
transition predominated in domains I, III, V, and VI. In addition to structural elements and motifs typical for group IIB
introns, several Allium-specific micro- and macrostructural mutations were revealed. A 290-bp deletion involving domains III and IV and part of domain
V was observed in A. altaicum, A. fistulosum, and A. schoenoprasum. Several indels and nucleotide substitutions were found to cause a deviation of the pre-mRNA secondary structure from the
consensus model of group II introns. 相似文献
18.
To overexpress the chitosanase gene (csn) in F. solani, a vector based on pCAMBIA 1300 was constructed. The csn gene, which is under control of the Aspergillus nidulans
gpdA promoter and A. nidulans trpC terminator, was introduced back into the F. solani genome by Agrobacterium tumefaciens-mediated transformation, and the herbicide-resistance gene bar from Streptomyces hygroscopicus was used as the selection marker. Transformants which showed a significant increase in chitosanase production (~2.1-fold
than control) were obtained. Southern blot analysis indicated that most transformants had a single-copy T-DNA integration. 相似文献
19.
Hiroyoshi Kubo 《Mycoscience》2009,50(5):400-406
Pilobolus crystallinus shows unique photoresponses at various growing stages. cDNAs for putative photoreceptors were cloned from this fungus. Three
genes named pcmada1, pcmada2, and pcmada3 were identified from the PCR fragments, and amplified with degenerated primers for the LOV domain, which is conserved in
many blue-light receptors. Deduced amino acid sequences for PCMADA1, PCMADA2, and PCMADA3 had one light-oxygen-voltage (LOV)-sensing
and two PER-ARNT-SIM (PAS) domains. A zinc finger DNA-binding motif was conserved in the C-terminals of PCMADA1 and PCMADA3.
However, PCMADA2 lacked the zinc finger motif. Expression of pcmada1 was suppressed by blue light whereas that of pcmada3 was promoted by blue-light irradiation. 相似文献