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1.
P. J. Punt I. A. van Gemeren J. Drint-Kuijvenhoven J. G. M. Hessing G. M. van Muijlwijk-Harteveld A. Beijersbergen C. T. Verrips C. A. M. J. J. van den Hondel 《Applied microbiology and biotechnology》1998,50(4):447-454
The function of the endoplasmic-reticulum-localized chaperone binding protein (BiP) in relation to protein secretion in filamentous
fungi was studied. It was shown that the overproduction of several homologous and heterologous recombinant proteins by Aspergillus strains induces the expression of bipA, the BiP-encoding gene from Aspergillus niger and Aspergillus awamori. As this result could imply that BiP plays a role in protein overproduction, the effect of modulation of bipA gene expression on protein secretion was studied in several recombinant strains expressing glucoamylase (glaA) fusion genes. For overproduction of BiPA in these strains, extra copies of the bipA gene under the control of an inducible promoter were introduced. To allow analysis of the effect of a decreased bipA expression level on protein secretion, replacement of the wild-type gene for a bipA gene driven by the glaA promoter was attempted. However, this endeavour failed because of the lethality of this replacement. Although the final amount
of secreted recombinant protein did not change significantly in strains with increased BiPA levels, increased levels of unprocessed
fusion protein were detected in the total protein extracts of these strains.
Received: 9 February 1998 / Received last revision: 26 May 1998 / Accepted: 14 June 1998 相似文献
2.
The anaerobe Eubacterium acidaminophilum has been shown to contain an uncharacterized peroxidase, which may serve to protect the sensitive selenoproteins in that
organism. We purified this peroxidase and found that it was identical with the substrate-specific “protein B”-complex of glycine
reductase. The “protein B”-complex consists of the selenocysteine-containing GrdB subunit and two subunits, which derive from
the GrdE proprotein. The specific peroxidase activity was 1.7 U (mg protein)−1 with DTT and cumene hydroperoxide as substrates. Immunoprecipitation experiments revealed that GrdB was important for DTT-
and NADH-dependent peroxidase activities in crude extracts, whereas the selenoperoxiredoxin PrxU could be depleted without
affecting these peroxidase activities. GrdB could be heterologously produced in Escherichia coli with coexpression of selB and selC from E. acidaminophilum for selenocysteine insertion. Although GrdB was sensitive to proteolysis, some full-size protein was present which accounted
for a peroxidase activity of about 0.5 U (mg protein)−1 in these extracts. Mutation of the potentially redox-active UxxCxxC motif in GrdB resulted in still significant, but decreased
activity. Heterologous GrdB was protected from degradation by full-length GrdE or by GrdE-domains. The GrdB-GrdE interaction
was confirmed by copurification of GrdE with Strep-tagged GrdB. The data suggest that GrdE domains serve to stabilise GrdB.
Dedicated to Prof. Dr. Gerhard Gottschalk. 相似文献
3.
Kikuchi T 《Amino acids》2008,35(3):541-549
It is well-known that the IgG-binding domain from staphylococcal protein A folds into a 3α helix bundle structure, while the
IgG-binding domain of streptococcal protein G forms an (α + β) structure. Recently, He et al. (Biochemistry 44:14055–14061,
2005) made mutants of these proteins from the wild types of protein A and protein G strains. These mutants are referred to as
protein A219 and protein G311, and it was showed that these two mutants have different 3D structures, i.e., the 3α helix bundle
structure and the (α + β) structure, respectively, despite the high sequence identity (59%). The purpose of our study was
to clarify how such 3D structural differences are coded in the sequences with high homology. To address this problem, we introduce
a predicted contact map constructed based on the interresidue average-distance statistics for prediction of folding properties
of a protein. We refer to this map as an average distance map (ADM). Furthermore, the statistics of interresidue distances
can be converted to an effective interresidue potential. We calculated the contact frequency of each residue of a protein
in random conformations with this effective interresidue potential, and then we obtained values similar to ϕ values. We refer
to this contact frequency of each residue as a p(μ) value. The comparison of the p(μ) values to the ϕ values for a protein suggests that p(μ) values reveal the information on the folding initiation site. Using these techniques, we try to extract the information
on the difference in the 3D structures of protein A219 and protein G311 coded in their amino acid sequences in the present
work. The results show that the ADM analyses and the p(μ) value analyses predict the information of folding initiation sites, which can be used to detect the 3D difference in both
proteins. 相似文献
4.
By using an oligonucleotide mixture corresponding to a region highly conserved among alternative sigma factors we identified
a new σ factor gene (rpoH) from Rhodobacter capsulatus. This gene encodes a protein of 34 kDa with strong similarity to the RpoH (σ
32) factors from other bacterial species. It was not possible to inactivate the R. capsulatusrpoH gene by introducing a resistance cassette, implying that it is essential for growth. The 5′ ends of the mRNAs were mapped
to two sequences with similarity to an rpoH- and an rpoD-dependent promoter, respectively. The amounts of both these mRNAs increased after heat shock, but were unaffected by a decrease
in oxygen tension. Western analysis using a σ factor-specific antibody revealed the accumulation of a protein of about 34 kDa after heat shock, and an increase in the
amounts of a protein with the same size after reduction of oxygen tension in R. capsulatus cultures.
Received: 16 March 1998 / Accepted: 28 July 1998 相似文献
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7.
C. P. J. Maury 《Origins of life and evolution of the biosphere》2009,39(2):141-150
The idea is advanced that under the extreme earth conditions for ~3.9 billions years ago, protein-based β-sheet molecular structures were the first self-propagating and information-processing biomolecules that evolved. The amyloid
structure of these aggregates provided an effective protection against the harsh conditions known to decompose both polyribonucleotides
and natively folded polypeptides. In the prebiotic amyloid world, both the replicative and informational functions were carried
out by structurally stable β-sheet protein aggregates in a prion-like mode involving templated self-propagation and storage of information in the β-sheet conformation. In this amyloid (protein)-first, hybrid replication-metabolism view, the synthesis of RNA, and the evolvement
of an RNA-protein world, were later, but necessary events for further biomolecular evolution to occur. I further argue that
in our contemporary DNA↔RNA→protein world, the primordial β-conformation-based information system is preserved in the form of a cytoplasmic epigenetic memory. 相似文献
8.
Adachi T Ito J Kawata K Kaya M Ishida H Sahara H Hata Y Ogino C Fukuda H Kondo A 《Applied microbiology and biotechnology》2008,81(4):711-719
A novel cell-surface display system was constructed in Aspergillus oryzae. Each of the five genes encoding the putative cell-wall-localized protein from the A. oryzae genome was cloned and these cell-surface anchor functions were examined by fusion to the C-terminal of the green fluorescent
protein (GFP). Using the MP1 and CWP proteins as anchor proteins, GFP signals were strongly observed on the cell surface of
recombinant A. oryzae. When these proteins were used as anchor proteins for cell-surface display of β-glucosidase from A. oryzae, enzyme activity was detected on the cell surface. In particular, β-glucosidase activity of recombinant A. oryzae using MP1, a putative glycosylphosphatidylinositol (GPI) anchor protein was higher than CWP. Based on these results, it was
concluded that the MP1 protein can act as a GPI-anchor protein in A. oryzae, and the proposed cell-surface display system using MP1 allows for the display of heterogeneous and endogenous proteins. 相似文献
9.
Atsushi Tanaka Hisashi Hirano Masahiro Kikuchi Shigeru Kitayama Hiroshi Watanabe 《Radiation and environmental biophysics》1996,35(2):95-99
In order to examine radiation-induced proteins in an extremely radioresistant bacterium, Deinococcus radiodurans R
1, changes in cellular proteins after γ-irradiation were analysed by two-dimensional gel electrophoresis and silver staining. Nine proteins (190, 120, 87, 60, 58,
52, 46, 41 and 41 kDa) were increased (or appeared) and more than 13 proteins diminished after γ-irradiation at 6 kGy. Increase of eight proteins (except for 190-kDa protein) was prevented when the cells were irradiated
in the presence of chloramphenicol. Three proteins, 87, 60 and 46 kDa, continued to be synthesized during post-irradiation
incubation, and the amounts of these proteins increased with higher doses in a range of 1 – 12 kGy. Changes in the amount
of proteins after irradiation in the R
1 strain were compared with those in a moderately radioresistant mutant (rec 1) and in a highly radiosensitive mutant (rec30). These three proteins were increased in both R
1 and rec 1, but not in rec 30, suggesting that they are characteristic for radioresistant strains. In addition, from the microsequence analysis, the 46-kDa
protein was found to be homologous to the EF-Tu protein of Escherichia coli, whereas the remarkable homologous sequence to the N-terminal of the 60-kDa protein was not found among the known proteins.
Received: 28 March 1995 / Accepted in revised form: 16 January 1996 相似文献
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11.
Haiyan Wei Xuhua Mi Ling Ji Lichuan Yang Qingjie Xia Yuquan Wei Isamu Miyamori ChunYuan Fan 《Biochemistry. Biokhimii?a》2010,75(3):304-309
In this study, we focused on the relationship between aldosterone and NOX1 expression in vascular smooth muscle cells (VSMCs). For the first time, with the use of specific inhibitors of protein kinase
C (PKC), we report that PKCδ mediates upregulation of NOX1 induced by 10 nM aldosterone in cultured VSMCs. Participation of PKC in the mediation of NOX1 regulation was further confirmed by the effect of diacylglycerol, a PKC agonist, on the NOX1 RNA in A7r5 cells with Northern blot analysis. To establish cause and effect, we next silenced the PKCδ gene partly by RNA
interference and found knockdown of PKCδ gene attenuated aldosterone-induced NOX1 expression, generation of superoxide, as well as protein synthesis in VSMCs. Taken together, these data indicated PKCδ might
mediate aldosterone-dependent NOX1 upregulation in VSMCs. In addition, we showed that the cascade from aldosterone to PKCδ activation had the participation
of the mineralocorticoid receptor. 相似文献
12.
M. V. Padkina L. V. Parfenova A. E. Gradoboeva E. V. Sambuk 《Applied Biochemistry and Microbiology》2010,46(4):409-414
The HuIFNA16, HuIFNB1, and BoIFNG genes encoding human α16, β-interferons and bovine γ-interferon were cloned under the control of the yeast Pichia pastoris AOX1 gene promoter. The yeast strains producing heterologous interferons intracellularly and extracellularly were constructed.
There was no effect of high level of heterologous protein synthesis on the yeast P. pastoris cell growth, unlike yeast Saccharomyces cerevisiae. The considerable part of the heterologous interferons was detected in the yeast P. pastoris soluble protein fraction but not in the “inclusion bodies.” The treatment of human β-interferon with endoglycosidase H showed
that protein was expressed in glycosylated and unglycosylated forms. On the strength of these data, the hypothesis was suggested
that the more effective heterologous gene expression in yeast P. pastoris and enhanced resistance of the methylotrophic yeast to negative effects of recombinant proteins was due to the special features
of its metabolism. 相似文献
13.
Makoto Takano Hiromi Kajiya-Kanegae Hideyuki Funatsuki Shoshi Kikuchi 《Molecular & general genetics : MGG》1998,260(4):388-394
We have isolated five cDNA clones (osk1–5) for protein kinases from rice which are related to SNF1 protein kinase of Saccharomyces cerevisiae. Based on the sequence homology, these cDNAs can be classified into two groups, group 1 (osk1) and group 2 (osk2–5). The products of these genes were demonstrated to be functional SNF1-related protein kinases by in vitro and in vivo experiments.
Recombinant proteins expressed from both groups of genes were fully active as protein kinases and could phosphorylate SAMS
peptide, a substrate specific for the SNF1/AMPK family, as well as themselves (autophosphorylation). Moreover, expression
of osk3 cDNA in yeast snf1 mutants restored SNF1 function. Northern blot analyses showed differential expression of these two gene groups; group 1 is expressed uniformly
in growing tissues (young roots, young shoots, flowers, and immature seeds), whereas group 2 is strongly expressed in immature
seeds. SNF1-related protein kinases have been reported from different plant species, such as rye, barley, Arabidopsis, tobacco, and potato, while the type of gene strongly expressed in immature seeds is known only in cereals such as rye, barley,
and, from our findings, in rice. Expression levels of the group 2 genes were further analyzed in seeds during seed maturation.
Expression is transiently increased in the early stages of seed maturation and then decreases. The expression peak precedes
those of the sbe1 and waxy genes, which are involved in starch synthesis in rice. Taken together, these findings suggest that group 2 OSK genes play important roles in the early stages of endosperm development in rice seeds.
Received: 30 April 1998 / Accepted: 20 August 1998 相似文献
14.
Brotosudarmo TH Mackowski S Hofmann E Hiller RG Bräuchle C Scheer H 《Photosynthesis research》2008,95(2-3):247-252
Peridinin–chlorophyll–protein (PCP), containing differently absorbing chlorophyll derivatives, are good models with which
to study energy transfer among monomeric chlorophylls (Chls) by both bulk and single-molecule spectroscopy. They can be obtained
by reconstituting the N-terminal domain of the protein (N-PCP) with peridinin and chlorophyll mixtures. Upon dimerization
of these “half-mers”, homo- and heterochlorophyllous complexes are generated, that correspond structurally to monomeric protomers
of native PCP from Amphidinium carterae. Heterochlorophyllous complexes contain two different Chls in the two halves of the complete structure. Here, we report reconstitution
of N-PCP with binary mixtures of Chl a, Chl b, and [3-acetyl]-Chl a. The ratios of the pigments were varied in the reconstitution mixture, and relative binding constants were determined from
quantification of these pigments in the reconstituted PCPs. We find higher affinities for both Chl b and [3-acetyl]-Chl a than for the native pigment, Chl a. 相似文献
15.
Ahmed Haouz Charles Twist Christian Zentz Patrick Tauc B. Alpert 《European biophysics journal : EBJ》1998,27(1):19-25
The catalytic oxidation of β-D-glucose by the enzyme glucose oxidase involves a redox change of the flavin coenzyme. The structure and the dynamics of
the two extreme glucose oxidase forms were studied by using infrared absorption spectroscopy of the amide I′ band, tryptophan
fluorescence quenching and hydrogen isotopic exchange. The conversion of FAD to FADH2 does not change the amount of α-helix present in the protein outer shell, but reorganises a fraction of random coil to β-sheet structure. The dynamics of the protein interior vary with the redox states of the flavin without affecting the motions
of the structural elements near the protein surface. From the structure of glucose oxidase given by X-ray crystallography,
these results suggest that the dynamics of the interface between the two monomers are involved in the catalytic mechanism.
Received: 27 December 1996 / Accepted: 18 July 1997 相似文献
16.
A series of ts mutations in the GSP1 gene of Saccharomyces cerevisiae was isolated by error-prone PCR. A total of 25 ts gsp1 strains was obtained. Each of these mutants showed between one and seven different amino acid alterations. In several of
these ts gsp1 strains, the same amino acid residues in Gsp1p were repeatedly mutated, indicating that our screen for ts gsp1 mutations was saturating. All of the ts gsp1 strains isolated had a defect in nuclear protein import, but only 16 of the 25 ts gsp1 strains had a defect in mRNA export. Thus, Gsp1p is suggested to be directly involved in nuclear protein import, but not
in mRNA export. Following release from α-factor arrest, 11 of the ts gsp1 mutants arrested in G1; the remainder did not show any specific cell-cycle arrest, at 37° C, the nonpermissive temperature.
While the mutants that are defective in both mRNA export and protein import have a tendency to arrest in G1, there was no
clear correlation between the cell cycle phenotype and the defects in mRNA export and nuclear protein import. Based on this,
we assume that Ran/Gsp1p GTPase regulates the cell cycle and the nucleus/cytosol exchange of macromolecules through interactions
with effectors that were independent of each other, and are differentially affected by mutation.
Received: 30 June 1997 / Accepted: 23 October 1997 相似文献
17.
Mitsuhiro Takeda Akira M. Ono Tsutomu Terauchi Masatsune Kainosho 《Journal of biomolecular NMR》2010,46(1):45-49
The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure
determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for
aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (ε- and ζ-SAIL Phe) and tyrosine
(ε-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic
ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized δ-SAIL Phe and δ-SAIL Tyr, which
allow us to observe and assign δ-13C/1H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the δ-,
ε- or ζ-13C/1H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the δ-, ε-, and ζ-proton
signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and
SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly 13C, 15N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of ζ-SAIL Phe and ε-SAIL Tyr would be practically the best choice
for protein structural determinations. 相似文献
18.
Hermansyah Walter A. Laviña Minetaka Sugiyama Yoshinobu Kaneko Satoshi Harashima 《Archives of microbiology》2010,192(3):157-165
The double disruptant of the S. cerevisiae protein phosphatase (PPase) genes, PTP2 (phosphotyrosine-specific PPase) and MSG5 (phosphotyrosine and phosphothreonine/serine-PPase) causes calcium-sensitive growth (Cas). Previous study using Fluorescent-activated cell sorting (FACS) analysis showed that this growth defect with calcium occurs
at G1–S transition in the cell cycle. We discovered that six non-essential protein kinase (PKase) disruptions (Δbck1, Δmkk1, Δslt2/Δmpk1, Δmck1, Δssk2 and Δyak1) suppressed the Cas-phenotype of the Δptp2 Δmsg5 double disruptant. Bck1p, Mkk1p and Slt2p are components of the mitogen-activated protein kinase (MAPK) cascade of cell wall
integrity pathway (Slt2 pathway), and Mck1p is its down regulator. Ssk2p is the MAPK kinase kinase of the high-osmolarity
glycerol (HOG) pathway, while Yak1p is a negative regulator for the cAMP-dependent PKA pathway. FACS analysis revealed that
only the disruption of Δssk2 and Δyak1 but not Δbck1, Δmkk1, Δslt2 and Δmck1 was able to suppress the delayed G1–S transition, suggesting that suppression of the growth defect is not always accompanied
by suppression of the G1–S transition delay. The discovery of these PKases as suppressors revealed that in addition to the
previously anticipated Slt2 pathway, HOG, Yak1p and Mck1p regulatory pathways may also be involved in the calcium sensitivity of the Δptp2 Δmsg5 double disruptant. 相似文献
19.
20.
N. Michel-Reydellet N. Desnoues M. de Zamaroczy C. Elmerich P. A. Kaminski 《Molecular & general genetics : MGG》1998,258(6):671-677
This work reports the characterisation of the Azorhizobium caulinodans amtB gene, the deduced protein sequence of which shares similarity to those of several ammonium transporters. amtB is located downstream from glnK, a glnB-like gene. It is cotranscribed with glnK from an NtrC- and σ54-dependent promoter. glnK and amtB insertion mutant strains have been isolated. Methylammonium uptake was assayed in these strains and in other mutant strains
in which the regulation of nitrogen metabolism is impaired. Our data suggest that the AmtB protein is an ammonium transporter,
which is mainly regulated by NtrC in response to nitrogen availability.
Received: 2 February 1998 / Accepted: 20 March 1998 相似文献