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1.
Axel Freischmidt Johannes Hiltl Hans Robert Kalbitzer Gudrun Horn-Katting 《Biotechnology letters》2013,35(3):389-395
Cell-free synthesis of recombinant proteins has emerged as an alternative method of protein production although protein yields still cannot compete with in vivo expression techniques. In systems based on S30 extracts of Escherichia coli unfavorable side-reactions are involved in limiting protein yields. Therefore, carrying out cell-free reactions at lower temperatures might be beneficial as side reactions should be decreased. In this study we show that by using the 5′-untranslated region of the cold-shock gene cspA from E. coli as mRNA leader in cell-free reactions, the expression temperature can be decreased and simultaneously leads to an increase in protein yields. A compensation for the lower activity of T7 RNA polymerase at lower temperatures enhances protein synthesis even further. Additionally, this 5′-untranslated region also standardizes the optimal expression temperature of different proteins. 相似文献
2.
When 50 S subunits from Escherichia coli are incubated in the presence of 4.3 m-LiCl, the resulting 4.3c core particle quantitatively lacks L20 in addition to other proteins. The 4.3c core can be reconstituted to an active 50 S subunit in the presence of total 50 S proteins by means of the second step incubation of the two-step reconstitution procedure. This finding indicates that the conformation of the 4.3c core is at least equivalent to the conformation of the reconstitution intermediate RI501(1) particle, which is exclusively formed in the first-step incubation. It follows that L20 is not necessary for the maintenance of the 4.3c core conformation. In contrast, the total reconstitution of an active 50 S particle from (23 S + 5 S) RNA and a protein preparation lacking L20 was fully dependent on the addition of L20. However, when the 4.3c core, which does not contain L20, is reconstituted with the same protein fraction, the activity of the resulting particle did not depend on the presence of L20. Thus, L20 is essential for the early assembly (occurring in the first-step incubation) but plays no role either in the late assembly steps, or the functions of the mature 50 S particle.Heat treatment of the 4.3c core distorts the 4.3c core conformation and leads to particles with lower s values. The degradation of the 4.3c core conformation is reduced when L20 is added. A further stabilization is obtained by the addition of (L20 + L24). Thus, L20 is dispensable for the maintenance of the 4.3c core conformation, but stabilizes this conformation. 相似文献
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A variant form of Escherichia coli ribosomal protein L7/L12 that lacked residues 42 to 52 (L7/L12 Δ42–52) in the hinge region was shown previously to be completely inactive in supporting polyphenylalanine synthesis although it bound to L7/L12 deficient core particles with the normal stoichiometry of four copies per particle (Oleinikov AV, Perroud B, Wang B, Traut RR (1993) J Biol Chem, 268, 917–922). The result suggested that the hinge confers flexibility that is required for activity because the resulting bent conformation allows the distal C-terminal domain to occupy a location on the body of the large ribosomal subunit proximal to the base of the L7/L12 stalk where elongation factors bind. Factor binding to the hinge-truncated variant was tested. As an alternative strategy to deleting residues from the hinge, seven amino acid residues within the putative hinge region were replaced by seven consecutive proline residues in an attempt to confer increased rigidity that might reduce or eliminate the bending of the molecule inferred to be functionally important. This variant, L7/L12: (Pro)7, remained fully active in protein synthesis. Whereas the binding of both factors in ribosomes containing L7/L12:Δ42–52 was decreased by about 50%, there was no loss of factor binding in ribosomes containing L7/L12:(Pro)7, as predicted from the retention of protein synthesis activity. The factor:ribosome complexes that contained L7/L12:Δ42–52 had the same low level of GTP hydrolysis as the core particles completely lacking L7/L12 and EF-G did not support translocation measured by the reaction of phe-tRNA bounds in hr Asite with puromycin. It is concluded that the hinge region is required for the functionally productive binding of elongation factors, and the defect in protein synthesis reported previously is due to this defect. The variant produced by the introduction of the putative rigid Pro7 sequence retains sufficient flexibility for full activity. 相似文献
5.
Some structural and functional properties of ribosomes from the hydrogen-oxidizing bacterium Alcaligenes eutrophus were studied in order to investigate the background of expression of genetic information at the translational level. Ribosomal proteins from 30S subunits of A. eutrophus H16 were separated by two-dimensional gel electrophoresis into 21 spots, those from 50S subunits into 32 spots. While electrophoretic mobilities of several ribosomal proteins differed markedly from those of Escherichia coli, proteins sharing common immunological determinants with E. coli ribosomal proteins S1 and L7/L12 were found in A. eutrophus. Shifting from heterotrophic to autotrophic conditions of growth had no influence on the ribosomal protein pattern. Ribosomes of A. eutrophus had similar requirements for Mg2+ and poly(U) concentrations for optimum polyphenylalanine synthesis as those of E. coli. Protein synthesis elongation factors Tu from A. eutrophus and E. coli were immunologically similar. Efficiency of the A. eutrophus polyphenylalanine-synthesizing system was comparable to that of an analogous system derived from E. coli. This suggests that A. eutrophus could be employed for efficient expression of recombinant DNA. 相似文献
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Antibodies prepared in rabbits against Escherichia coli ribosomal proteins L7/L12 are reported to be immunologically cross-reactive with some ribosomal proteins on the 60 S subunit of eukaryote ribosomes (Wool & Stöffler, 1974; Stöffler et al., 1974). We have confirmed these reports and extended this finding to a detailed study of the functional properties of eukaryote ribosomes which are affected by these cross-reacting antibodies. We report here the partial reactions in protein synthesis that are inhibited by the anti-L7/L12 IgG (immunoglobulin G) preparations using a chicken liver system. The following reactions were inhibited: EF-1 (elongation factor 1) dependent binding of aminoacyl-tRNA to ribosomes and GTP hydrolysis; EF-2 dependent binding of nucleotide to ribosomes and GTP hydrolysis; binding of [14C]ADP-ribosyl · EF-2 to ribosomes. This last reaction is more sensitive to the antibody inhibition than the corresponding nucleotide binding reaction. We show that the inhibitions were not simply non-specific precipitation of ribosomes by IgG, in that monovalent Fabs were also inhibitory, and peptidyl transferase activity was not inhibited. The functions inhibited with the IgG preparations in the chicken liver system are analogous to those inhibited in the homologous E. coli system. Thus the cross-reacting protein is functionally as well as immunologically conserved. 相似文献
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Interaction among silkworm ribosomal proteins P1, P2 and P0 required for functional protein binding to the GTPase-associated domain of 28S rRNA 总被引:5,自引:0,他引:5
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Shimizu T Nakagaki M Nishi Y Kobayashi Y Hachimori A Uchiumi T 《Nucleic acids research》2002,30(12):2620-2627
Acidic ribosomal phosphoproteins P0, P1 and P2 were isolated in soluble form from silkworm ribosomes and tested for their interactions with each other and with RNA fragments corresponding to the GTPase-associated domain of residues 1030–1127 (Escherichia coli numbering) in silkworm 28S rRNA in vitro. Mixing of P1 and P2 formed the P1–P2 heterodimer, as demonstrated by gel mobility shift and chemical crosslinking. This heterodimer, but neither P1 or P2 alone, tightly bound to P0 and formed a pentameric complex, presumably as P0(P1–P2)2, assumed from its molecular weight derived from sedimentation analysis. Complex formation strongly stimulated binding of P0 to the GTPase-associated RNA domain. The protein complex and eL12 (E.coli L11-type), which cross-bound to the E.coli equivalent RNA domain, were tested for their function by replacing with the E.coli counterparts L10.L7/L12 complex and L11 on the rRNA domain within the 50S subunits. Both P1 and P2, together with P0 and eL12, were required to activate ribosomes in polyphenylalanine synthesis dependent on eucaryotic elongation factors as well as eEF-2-dependent GTPase activity. The results suggest that formation of the P1–P2 heterodimer is required for subsequent formation of the P0(P1–P2)2 complex and its functional rRNA binding in silkworm ribosomes. 相似文献
10.
Wai Kit Ma Rachel Hendrix Claire Stewart Eric V. Campbell Mitchell Lavarias Kolyn Morris Shauna Nichol Matthew J. Gage 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(4):808-816
Intrinsically disordered proteins (IDPs) are a unique class of proteins that do not require a stable structure for function. The importance of IDPs in many biological processes has been established but there remain unanswered questions about their evolution and conservation of their disordered state within a protein family. Our group has been studying the structural similarities among orthologous FlgM proteins, a model class of IDPs. We have previously shown that the FlgM protein from the thermophile Aquifex aeolicus has more structure at A. aeolicus' physiological temperature (85 °C) than is observed for the Salmonella typhimurium FlgM, suggesting that the disordered nature of FlgM varies among organisms and is not universally conserved. In this work, we extend these studies to the FlgM proteins from Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, and Bacillus subtilis. We demonstrate that the B. subtilis, E. coli, and S. typhimurium FlgMs exist in a premolten globule-like conformation, though the B. subtilis FlgM is in a more compacted conformation than the other two. The P. aeruginosa and P. mirabilis FlgM proteins exist in a currently unknown conformation that is not either coil-like or premolten globule-like. The P. aeruginosa FlgM appears to contain more weak intramolecular contacts given its more compacted state than the P. mirabilis FlgM. These results provide experimental evidence that members of the same protein family can exhibit different degrees of disorder, though understanding how different disordered states evolve in the same protein family will require more study. 相似文献
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70 S ribosomes from Escherichia coli have been reacted with the bifunctional reagent 1,4-phenyldiglyoxal under near physiological conditions. As a result of the cross-linking reaction a number of high-molecular-weight protein fractions with altered electrophoretic mobility could be isolated. A new chemical procedure has been introduced to reverse the cross-links between proteins at least partially. The cleavage reaction did not affect the gel electrophoretic mobility of the proteins. Thus a direct identification of cross-linked proteins using one- or two-dimensional gels was made possible. Two protein trimers, S3-S4-S5 and L1-S4-S5, as well as five protein dimers, S3-S4, L6-L7/12, L10-L7/12, S9-L19 and L18-L19 could be identified as close neighbours in the E. coli 70 S ribosome. The protein pairs S9-L19 and L18-L19 had previously not been identified as near neighbours using cross-linking studies. 相似文献
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Chang-Ho Kang YuJin Shin YongGyeong Kim Jae-Seong So 《Biotechnology and Bioprocess Engineering》2016,21(1):46-52
Microorganisms intended for use as probiotics in aquaculture should exert antimicrobial activity and be regarded as safe not only for their aquatic hosts but also for their surrounding environments and humans. The objective of this work was to investigate antimicrobial activity against various pathogens, bile salt tolerance, and acid tolerance of 65 presumptive Lactobacillus spp. isolated from shellfish samples. Four strains (HL1, HL12, HL20, and JL28) were selected after qualitatively identifying high levels of antimicrobial activity against bacteria including Staphylococcus aureus, Salmonella typhimurium, Salmonella enteritidis, Escherichia coli O157:H7, Vibrio ichthyoenteri, Edwardsiella tarda, Streptococcus iniae, and V. parahaemolyticus. The sequence analysis of their 16S rRNA genes revealed that the four strains belong to the Lactobacillus plantarum species. In addition, their survivability was tested in bile salt and acidic conditions to show their potential use as probiotics in the gastrointestinal tract. 相似文献
13.
Karel Mikulík Ivan Janda Jaroslav Weiser Anna Jirov 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,699(3):203-210
Three different two-dimensional polyacrylamide gel electrophoretic systems were employed for identification of individual ribosomal proteins of Streptomyces aureofaciens. Proteins of small subunits were resolved into 21 spots. Larger ribosomal subunits contained 35 proteins. The separated ribosomal proteins from 50 S subunits were transferred on nitrocellulose membranes for immunochemical estimations. Antibodies developed against 50 S proteins of S. aureofaciens and Escherichia coli were used for identification of structural homologies between 50 S proteins of the two species. Results of the experiments indicate that about one half of the 50 S proteins of S. aureofaciens share common immunochemical determinants with corresponding proteins of 50 S subunits of E. coli. Evidence is presented that acidic ribosomal protein SL5 of large ribosomal subunits of S. aureofaciens can be assembled to E. coli P0 cores lacking proteins L7/L12. Reconstitution of the P0 cores with proteins SL5 or L7/L12 led to restoration of 78% activity in polyphenylalanine synthesis. 相似文献
14.
Stability of the 'L12 stalk' in ribosomes from mesophilic and (hyper)thermophilic Archaea and Bacteria
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The ribosomal stalk complex, consisting of one molecule of L10 and four or six molecules of L12, is attached to 23S rRNA via protein L10. This complex forms the so-called ‘L12 stalk’ on the 50S ribosomal subunit. Ribosomal protein L11 binds to the same region of 23S rRNA and is located at the base of the ‘L12 stalk’. The ‘L12 stalk’ plays a key role in the interaction of the ribosome with translation factors. In this study stalk complexes from mesophilic and (hyper)thermophilic species of the archaeal genus Methanococcus and from the Archaeon Sulfolobus solfataricus, as well as from the Bacteria Escherichia coli, Geobacillus stearothermophilus and Thermus thermophilus, were overproduced in E.coli and purified under non-denaturing conditions. Using filter-binding assays the affinities of the archaeal and bacterial complexes to their specific 23S rRNA target site were analyzed at different pH, ionic strength and temperature. Affinities of both archaeal and bacterial complexes for 23S rRNA vary by more than two orders of magnitude, correlating very well with the growth temperatures of the organisms. A cooperative effect of binding to 23S rRNA of protein L11 and the L10/L124 complex from mesophilic and thermophilic Archaea was shown to be temperature-dependent. 相似文献
15.
Takayuki Manabe Mayu KatoTakayuki Ueno Kiyoshi Kawasaki 《Biochemical and biophysical research communications》2013
Gram-negative bacteria, including Escherichia coli, release outer membrane vesicles (OMVs) that are derived from the bacterial outer membrane. OMVs contribute to bacterial cell–cell communications and host–microbe interactions by delivering components to locations outside the bacterial cell. In order to explore the molecular machinery involved in OMV biogenesis, the role of a major OMV protein was examined in the production of OMVs from E. coli W3110, which is a widely used standard E. coli K-12 strain. In addition to OmpC and OmpA, which are used as marker proteins for OMVs, an analysis of E. coli W3110 OMVs revealed that they also contain abundant levels of FliC, which is also known as flagellin. A membrane-impermeable biotin-labeling reagent did not label FliC in intact OMVs, but labeled FliC in sonically disrupted OMVs, suggesting that FliC is localized in the lumen of OMV. Compared to the parental strain expressing wild-type fliC, an E. coli strain with a fliC-null mutation produced reduced amounts of OMVs based on both protein and phosphate levels. In addition, an E. coli W3110-derived strain with a null-mutation in flgK, which encodes flagellar hook-associated protein that is essential along with FliC for flagella synthesis, also produced fewer OMVs than the parental strain. Taken together, these results indicate that the ability to form flagella, including the synthesis of flagella proteins, affects the production of E. coli W3110 OMVs. 相似文献
16.
Gloria Blanco M. Rosario Rodicio Anna Maria Puglia † Carmen Méndez Charles J. Thompson ‡ José A. Salas 《Molecular microbiology》1994,12(3):375-385
Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins being synthesized were pulse-labelled with [35 S]-methionine, separated by two-dimensional poly-acrylamide gel electrophoresis, and quantified using the Bioimage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coliβ-galactosldase. The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHl fragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10-L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E. coli and other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHl fragment. 相似文献
17.
S R Fahnestock 《Archives of biochemistry and biophysics》1977,180(2):555-561
Functional homology between the 50 S ribosomal protein L3 from Bacillus stearothermophilus and a protein from each of three other species of Bacillaceae (Bacillus licheniformis, Bacillus megaterium, and Bacillus subtilis) is demonstrated by substituting each of the proteins for B. stearothermophilus L3 in active reconstituted ribosomes. The structurally related protein from Escherichia coli, L2, cannot replace B, stearothermophilus L3, nor can any other E. coli protein. 相似文献
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Molecular Cloning, Sequencing, and Expression of a Chemoreceptor Gene from Leptospirillum ferrooxidans
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We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacterium Leptospirillum ferrooxidans. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillum chemotaxis receptor I. This is the first sequence reported for a gene from L. ferrooxidans encoding a protein. The lcrI gene showed both ς28-like and ς70-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP from Escherichia coli), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed in E. coli, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in E. coli. This was most likely due to the fact that the periplasmic pH of E. coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain. 相似文献