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1.
GroELx and GroESx proteins of symbiotic X-bacteria from Amoeba proteus were overproduced in Escherichia coli transformed with pAJX91 and pUXGPRM, respectively, and their chaperonin functions were assayed. We utilized σ70-dependent specific promoters of groEx in the expression vectors and grew recombinant cells at 37°C to minimize coexpression of host groE of E. coli. For purifying the proteins, we applied the principle of heat stability for GroELx and pI difference for GroESx to minimize copurification with the hosts GroEL and GroES, respectively. After ultracentrifugation in a sucrose density gradient, the yield and purity of GroELx were 56 and 89%, respectively. The yield and purity of GroESx after anion-exchange chromatography were 62 and 91%, respectively. Purified GroELx had an ATPase activity of 53.2 nmol Pi released/min/mg protein at 37°C. The GroESx protein inhibited ATPase activity of GroELx to 60% of the control at a ratio of 1 for GroESx-7mer/GroELx-14mer. GroESLx helped refolding of urea-unfolded rhodanese up to 80% of the native activity at 37°C. By chemical cross-linking analysis, oligomeric properties of GroESx and GroELx were confirmed as GroESx7 and GroELx14 in two stacks of GroELx7. In this study, we developed a method for the purification of GroESLx and demonstrated that their chaperonin function is homologous to GroESL of E. coli.  相似文献   

2.
The thermostability of the recombinant alpha- and beta-subunit homo-oligomers (alpha16mer and beta16mer) and of natural chaperonins purified from cultured Thermococcus strain KS-1 cells was measured to understand the mechanism for the thermal acclimatization of T. KS-1. The beta-subunit content of the natural chaperonin from cells grown at 90 degrees C was higher than that at 80 degrees C. The optimum temperature for ATPase activity of the natural chaperonins was 80-90 degrees C, whereas that for alpha16mer and beta16mer was 60 degrees C and over 90 degrees C respectively. Judging from the ATPase activity, beta16mer was more thermostable than alpha16mer. The thermostabilities of the natural chaperonins were intermediate between alpha16mer and beta16mer, whereas the natural chaperonin with a higher beta-subunit content was more stable than that with a lower beta-subunit content. Native polyacrylamide gel electrophoresis (PAGE) revealed that the chaperonin oligomers thermally dissociated to their ATPase-inactive monomers. The thermal denaturation process monitored by circular dichroism showed that the free beta-subunit was more stable than the free alpha-subunit, and that the secondary structure of the chaperonin monomer in the oligomer was more stable than that in the free monomer. These results suggest that the structure of these subunits was stabilized in the oligomer, and that an increase in the beta-subunit content conferred higher thermostability to the natural hetero-oligomeric chaperonin.  相似文献   

3.
Over last two decades many researchers have demonstrated the mechanisms of how the Escherichia coli chaperonin GroEL and GroES work in the binding and folding of different aggregation prone substrate proteins both in vivo and in vitro. However, preliminary aspects, such as influence of co-expressing GroEL and GroES on the over expression of other recombinant proteins in E. coli cells and subsequent growth aspects, as well as the conditions for optimum production of recombinant proteins in presence of recombinant chaperones have not been properly investigated. In the present study we have demonstrated the temperature dependent growth characteristics of E. coli cells, which are over expressing recombinant aconitase and how the co-expression of E. coli chaperonin GroEL and GroES influence the growth rate of the cells and in vivo folding of recombinant aconitase. Presence of co-expressed GroEL reduces the aconitase over-expression drastically; however, exogenous GroEL & GroES together compensate this reduction. For the aconitase over-expressing cells the growth rate decreases by 30% at 25 degrees C when compared with the M15 E. coli cells, however, there is an increase of 20% at 37 degrees C indicating the participation of endogenous chaperonin in the folding of a fraction of over expressed aconitase. However, in presence of co-expressed GroEL and GroES the growth rate of aconitase producing cells was enhanced by 30% at 37 degrees C confirming the assistance of exogenous chaperone system for the folding of recombinant aconitase. Optimum in vivo folding of aconitase requires co-production of complete E. coli chaperonin machinery GroEL and GroES together.  相似文献   

4.
The mutS gene, implicated in DNA mismatch repair, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence encoded a 819-amino acid protein with a molecular mass of 91.4 kDa. Its predicted amino acid sequence showed 56 and 39% homology with Escherichia coli MutS and human hMsh2 proteins, respectively. The T.thermophilus mutS gene complemented the hypermutability of the E.coli mutS mutant, suggesting that T.thermophilus MutS protein was active in E.coli and could interact with E.coli MutL and/or MutH proteins. The T.thermophilus mutS gene product was overproduced in E.coli and then purified to homogeneity. Its molecular mass was estimated to be 91 kDa by SDS-PAGE but approx. 330 kDa by size-exclusion chromatography, suggesting that T.thermophilus MutS protein was a tetramer in its native state. Circular dichroic measurements indicated that this protein had an alpha-helical content of approx. 50%, and that it was stable between pH 1.5 and 12 at 25 degree C and was stable up to 80 degree C at neutral pH. Thermus thermophilus MutS protein hydrolyzed ATP to ADP and Pi, and its activity was maximal at 80 degrees C. The kinetic parameters of the ATPase activity at 65 degrees C were Km = 130 microM and Kcat = 0.11 s(-1). Thermus thermophilus MutS protein bound specifically with G-T mismatched DNA even at 60 degrees C.  相似文献   

5.
The cpn60 and cpn10 genes from psychrophilic bacterium, Oleispira antarctica RB8, showed a positive effect in Escherichia coli growth at low temperature, shifting its theoretical minimal growth temperature from +7.5 degrees C to -13.7 degrees C [Ferrer, M., Chernikova, T.N., Yakimov, M., Golyshin, P.N., and Timmis, K.N. (2003) Nature Biotechnol 21: 1266-1267]. To provide experimental support for this finding, Cpn60 and 10 were overproduced in E. coli and purified to apparent homogeneity. Recombinant O.Cpn60 was identical to the native protein based on tetradecameric structure, and it dissociates during native PAGE. Gel filtration and native PAGE revealed that, in vivo and in vitro, (O.Cpn60)(7) was the active oligomer at 4-10 degrees C, whereas at > 10 degrees C, this complex was converted to (O.Cpn60)(14). The dissociation reduces the ATP consumption (energy-saving mechanism) and increases the refolding capacity at low temperatures. In order for this transition to occur, we demonstrated that K468 and S471 may play a key role in conforming the more advantageous oligomeric state in O.Cpn60. We have proved this hypothesis by showing that single and double mutations in K468 and S471 for T and G, as in E.GroEL, produced a more stable double-ring oligomer. The optimum temperature for ATPase and chaperone activity for the wild-type chaperonin was 24-28 degrees C and 4-18 degrees C, whereas that for the mutants was 45-55 degrees C and 14-36 degrees C respectively. The temperature inducing unfolding (T(M)) increased from 45 degrees C to more than 65 degrees C. In contrast, a single ring mutant, O.Cpn60(SR), with three amino acid substitutions (E461A, S463A and V464A) was as stable as the wild type but possessed refolding activity below 10 degrees C. Above 10 degrees C, this complex lost refolding capacity to the detriment of the double ring, which was not an efficient chaperone at 4 degrees C as the single ring variant. We demonstrated that expression of O.Cpn60(WT) and O.Cpn60(SR) leads to a higher growth of E. coli at 4 degrees C ( micro (max), 0.22 and 0.36 h(-1) respectively), whereas at 10-15 degrees C, only E. coli cells expressing O.Cpn60 or O.Cpn60(DR) grew better than parental cells (-cpn). These results clearly indicate that the single-to-double ring transition in Oleispira chaperonin is a wild-type mechanism for its thermal acclimation. Although previous studies have also reported single-to-double ring transitions under many circumstances, this is the first clear indication that single-ring chaperonins are necessary to support growth when the temperature falls from 37 degrees C to 4 degrees C.  相似文献   

6.
Escherichia coli MsbA, the proposed inner membrane lipid flippase, is an essential ATP-binding cassette transporter protein with homology to mammalian multidrug resistance proteins. Depletion or loss of function of MsbA results in the accumulation of lipopolysaccharide and phospholipids in the inner membrane of E. coli. MsbA modified with an N-terminal hexahistidine tag was overexpressed, solubilized with a nonionic detergent, and purified by nickel affinity chromatography to approximately 95% purity. The ATPase activity of the purified protein was stimulated by phospholipids. When reconstituted into liposomes prepared from E. coli phospholipids, MsbA displayed an apparent K(m) of 878 microm and a V(max) of 37 nmol/min/mg for ATP hydrolysis in the presence of 10 mm Mg(2+). Preincubation of MsbA-containing liposomes with 3-deoxy-d-mannooctulosonic acid (Kdo)(2)-lipid A increased the ATPase activity 4-5-fold, with half-maximal stimulation seen at 21 microm Kdo(2)-lipid A. Addition of Kdo(2)-lipid A increased the V(max) to 154 nmol/min/mg and decreased the K(m) to 379 microm. Stimulation was only seen with hexaacylated lipid A species and not with precursors, such as diacylated lipid X or tetraacylated lipid IV(A). MsbA containing the A270T substitution, which renders cells temperature-sensitive for growth and lipid export, displayed ATPase activity similar to that of the wild type protein at 30 degrees C but was significantly reduced at 42 degrees C. These results provide the first in vitro evidence that MsbA is a lipid-activated ATPase and that hexaacylated lipid A is an especially potent activator.  相似文献   

7.
Mendoza JA  Dulin P  Warren T 《Cryobiology》2000,41(4):319-323
The chaperonins GroEL and GroES were shown to facilitate the refolding of urea-unfolded rhodanese in an ATP-dependent process at 25 or 37 degrees C. A diminished chaperonin activity was observed at 10 degrees C, however. At low temperature, GroEL retains its ability to form a complex with urea-unfolded rhodanese or with GroES. GroEL is also able to bind ATP at 10 degrees C. Interestingly, the ATPase activity of GroEL was highly decreased at low temperatures. Hydrolysis of ATP by GroEL was 60% less at 10 degrees C than at 25 degrees C. We conclude that the reduced hydrolysis of ATP by GroEL is a major but perhaps not the only factor responsible for the diminished chaperonin activity at 10 degrees C. GroEL may function primarily at higher temperatures in which the ability of GroEL to hydrolyze ATP is not compromised.  相似文献   

8.
We have constructed a full-length cDNA that encodes soybean seed lipoxygenase L-1 and have expressed it in Escherichia coli. This gene was inserted into a pT7-7 expression vector, containing the T7 RNA polymerase promoter. E. coli, strain BL21 (DE3), which carries the T7 promoter in its genome, was transfected with the plasmid. Expression of this gene when the cells were cultured at 37 degrees C yielded polypeptide that was recognized by anti-L-1 antibody, but had very little lipoxygenase activity. Yields of active enzyme were markedly increased when cells were cultured at 15-20 degrees C. When ethanol, which has been reported to be an excellent elicitor of heat-shock proteins in E. coli, was also present at a level of 3% the yield was further increased by 40%. Under optimum conditions 22-30 mg of soluble active enzyme was obtained per liter of culture.  相似文献   

9.
Chromatium vinosum contains a polypeptide that is functionally and structurally similar to the Escherichia coli chaperonin 10. The protein has been purified to homogeneity by sucrose density gradient centrifugation followed by gel filtration using a Bio-Gel A-1.5 m column. The molecular mass of chaperonin 10, as determined by gel filtration or nondenaturing polyacrylamide gel electrophoresis, is 95 kDa. The oligomer is composed of seven or eight subunits. Comparisons of the overall amino acid composition and N-terminal sequences among chaperonin 10 species from C. vinosum and E. coli reflect a high degree of similarity. A physical association between chaperonins 60 and 10 from C. vinosum, in vitro, is supported by three experimental approaches. First, the proteins form a stable binary complex in sucrose density gradients, gel filtration chromatography, and nondenaturing polyacrylamide gel electrophoresis, solely in the presence of ATP and Mg2+. Second, chaperonin 10 from C. vinosum binds, selectively, to a chaperonin 60-coupled Affi-Gel 10 matrix column. Third, a slight molar excess of chaperonin 10 is able to abolish, almost completely, the ATPase in chaperonin 60. The rate for ATPase activity of chaperonin 60 from C. vinosum is enhanced when supplemented with monovalent cations.  相似文献   

10.
AIM: To evaluate the effect of NaCl, growth medium and temperature on the antimicrobial activity of bovine lactoferrin (LF) against Escherichia coli O157:H7 in the presence of different chelating agents. METHODS AND RESULTS: LF (32 mg ml(-1)) was tested against E. coli O157:H7 strain 3081 in Luria broth (LB) and All Purpose Tween (APT) broth with metal ion chelators sodium bicarbonate (SB), sodium lactate (SL), sodium hexametaphosphate (SHMP), ethylene diamine tetraacetic acid (EDTA) or quercetin at 0.5 and 2.5% NaCl at 10 and 37 degrees C. LF and the chelators were tested against four other E. coli O157:H7 strains in LB at 2.5% NaCl and 10 degrees C. LF alone was bacteriostatic against strains 3081 and LCDC 7283 but other strains grew. Antimicrobial effectiveness of LF was reduced in APT broth but enhanced by SB at 2.5% NaCl and 10 degrees C where 4.0 log(10) CFU ml(-1) inoculated cells were killed. EDTA enhanced antimicrobial action of the LF-SB combination. SL alone was effective against E. coli O157:H7 but a reduction in its activity at 2.5% NaCl and 10 degrees C was reversed by LF. The combinations LF-SHMP and LF-quercetin were more effective at 37 degrees C and NaCl effects varied. CONCLUSIONS: LF plus SB or SL were bactericidal toward the same 3/5 E. coli O157:H7 strains and inhibited growth of the others at 2.5% NaCl and 10 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of LF with either SL or SB shows potential for reducing viability of E. coli O157:H7 in food systems containing NaCl at reduced, but growth permissive temperature.  相似文献   

11.
Fate of enterohemorrhagic Escherichia coli O157:H7 in bovine feces.   总被引:12,自引:0,他引:12       下载免费PDF全文
G Wang  T Zhao    M P Doyle 《Applied microbiology》1996,62(7):2567-2570
Dairy cattle have been identified as a principal reservoir of Escherichia coli O157:H7. The fate of this pathogen in bovine feces at 5, 22, and 37 degrees C was determined. Two levels of inocula (10(3) and 10(5) CFU/g) of a mixture of five nalidixic acid-resistant E. coli O157:H7 strains were used. E. coli O157:H7 survived at 37 degrees C for 42 and 49 days with low and high inocula, respectively, and at 22 degrees C for 49 and 56 days with low and high inocula, respectively. Fecal samples at both temperatures had low moisture contents (about 10%) and water activities ( < 0.5) near the end of the study. E. coli O157:H7 at 5 degrees C survived for 63 to 70 days, with the moisture content (74%) of feces remaining high through the study. Chromosomal DNA fingerprinting of E. coli O157:H7 isolates surviving near the completion of the study revealed that the human isolate strain 932 was the only surviving strain at 22 or 37 degrees C. All five strains were isolated near the end of incubation from feces held at 5 degrees C. Isolates at each temperature were still capable of producing both verotoxin 1 and verotoxin 2. Results indicate that E. coli O157:H7 can survive in feces for a long period of time and retain its ability to produce verotoxins. Hence, bovine feces are a potential vehicle for transmitting E. coli O157:H7 to cattle, food, and the environment. Appropriate handling of bovine feces is important to control the spread of this pathogen.  相似文献   

12.
The cpkA gene encoding a second (alpha) subunit of archaeal chaperonin from Pyrococcus kodakaraensis KOD1 was cloned, sequenced, and expressed in Escherichia coli. Recombinant CpkA was studied for chaperonin functions in comparison with CpkB (beta subunit). The effect on decreasing the insoluble form of proteins was examined by coexpressing CpkA or CpkB with CobQ (cobyric acid synthase from P. kodakaraensis) in E. coli. The results indicate that both CpkA and CpkB effectively decrease the amount of the insoluble form of CobQ. Both CpkA and CpkB possessed the same ATPase activity as other bacterial and eukaryal chaperonins. The ATPase-deficient mutant proteins CpkA-D95K and CpkB-D95K were constructed by changing conserved Asp95 to Lys. Effect of the mutation on the ATPase activity and CobQ solubilization was examined. Neither mutant exhibited ATPase activity in vitro. Nevertheless, they decreased the amount of the insoluble form of CobQ by coexpression as did wild-type CpkA and CpkB. These results implied that both CpkA and CpkB could assist protein folding for nascent protein in E. coli without requiring energy from ATP hydrolysis.  相似文献   

13.
The effect of atmospheric composition and storage temperature on growth and survival of uninjured and sublethally heat-injured Escherichia coli O157:H7, inoculated onto brain heart infusion agar containing 0.3% beef extract (BEM), was determined. BEM plates were packaged in barrier bags in air, 100% CO2, 100% N2, 20% CO2: 80% N2, and vacuum and were stored at 4, 10, and 37 degrees C for up to 20 days. Package atmosphere and inoculum status (i.e., uninjured or heat-injured) influenced (P < 0.01) growth and survival of E. coli O157:H7 stored at all test temperatures. Growth of heat-injured E. coli O157:H7 was slower (P < 0.01) than uninjured E. coli O157:H7 stored at 37 degrees C. At 37 degrees C, uninjured E. coli O157:H7 reached stationary phase growth earlier than heat-injured populations. Uninjured E. coli O157:H7 grew during 10 days of storage at 10 degrees C, while heat-injured populations declined during 20 days of storage at 10 degrees C. Uninjured E. coli O157:H7 stored at 10 degrees C reached stationary phase growth within approximately 10 days in all packaging atmospheres except CO2. Populations of uninjured and heat-injured E. coli O157:H7 declined throughout storage for 20 days at 4 degrees C. Survival of uninjured populations stored at 4 degrees C, as well as heat-injured populations stored at 4 and 10 degrees C, was enhanced in CO2 atmosphere. Survival of heat-injured E. coli O157:H7 at 4 and 10 degrees C was not different (P > 0.05). Uninjured and heat-injured E. coli O157:H7 are able to survive at low temperatures in the modified atmospheres used in this study.  相似文献   

14.
The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.  相似文献   

15.
The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.  相似文献   

16.
Secretory production of human leptin in Escherichia coli   总被引:4,自引:0,他引:4  
Human leptin is a 16 kDa (146 amino acids) protein secreted from adipocytes and influences body weight homeostasis. In this study, human leptin was produced and secreted efficiently in Escherichia coli using a novel Bacillus sp. endoxylanase signal peptide. The endoxylanase signal sequence consisted of 28 amino acids (84 bp) was fused to the leptin structural gene. The fused gene was expressed using an inducible promoter (T7 or Trc) by adding 1 mM IPTG. Using T7 promoter in E. coli BL21(DE3), most of protein produced was in a premature form. Using the Trc promoter, which is weaker than T7, leptin was efficiently produced and secreted as a mature form (40% of total proteins) at 37 degrees C. However, most of leptin (about 90%) formed the inclusion bodies in the periplasmic space of E. coli. At 30 degrees C, ca. 90% of leptin was produced in a soluble form, but the total amount of leptin produced was 40% less than that obtained at 37 degrees C. When the periplasmic oxidoreductase of E. coli, DsbA, was co-expressed, 69% of the secreted leptin (26% of total proteins) was produced as soluble form at 37 degrees C without the decrease of the amount of leptin produced.  相似文献   

17.
AIMS: The objective of this study was to determine the combined effects of water activity (a(w)), chemical treatment and temperature on Salmonella and Escherichia coli O157:H7 inoculated onto alfalfa seeds. METHODS AND RESULTS: Alfalfa seeds inoculated with Salmonella or E. coli O157:H7 and adjusted to various a(w) values were subjected to simultaneous and separate treatments with chemicals and heat. The rate of death of both pathogens was correlated with increased a(w) (0.15-0.60) and temperature (5-37 degrees C) over a 52-week storage period. Higher seed a(w) enhanced the inactivation of pathogens on seeds heated at 50-70 degrees C for up to 24 h. Treatment of seeds with water, 1% Ca(OH)2, 1% Tween 80, 1% Ca(OH)2 plus 1% Tween 80 or 40 mg l(-1) Tsunami 200 at 23 or 55 degrees C for 2 min significantly (alpha=0.05) reduced populations of Salmonella and E. coli O157:H7. CONCLUSIONS: Overall, at the combinations of temperature and concentrations of chemicals tested, 1% Ca(OH)2 was most effective in killing Salmonella and E. coli O157:H7 without reducing seed viability. SIGNIFICANCE AND IMPACT OF THE STUDY: None of the treatments evaluated in this study, whether applied separately or in combination, eliminated Salmonella or E. coli O157:H7 on alfalfa seeds without sacrificing the viability of the seeds. It remains essential that practices to prevent the contamination of alfalfa seeds be strictly followed in order to minimize the risk of Salmonella and E. coli O157:H7 infections associated with sprouts produced from these seeds.  相似文献   

18.
K C Terlesky  F R Tabita 《Biochemistry》1991,30(33):8181-8186
Two heat-shock proteins that show high identity with the Escherichia coli chaperonin 60 (groEL) and chaperonin 10 (groES) chaperonin proteins were purified and characterized from photolithoautotrophically grown Rhodobacter sphaeroides. The proteins were purified by using sucrose density gradient centrifugation and Mono-Q anion-exchange chromatography. In the presence of 1 mM ATP, the chaperonin 10 and chaperonin 60 proteins bound to each other and comigrated as a large complex during sucrose density gradient centrifugation. The native molecular weights of each protein as determined by gel filtration chromatography were 889,200 for chaperonin 60 and 60,000 for chaperonin 10. Chaperonin 60 is comprised of monomers with a molecular weight of 61,000 and chaperonin 10 is comprised of monomers with a molecular weight of 12,700 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chaperonin 60 was 9.3% of the total soluble cell protein during photolithoautotrophic growth which increased to 28.5% following heat-shock treatment. When cells were grown photoheterotrophically or chemoheterotrophically, chaperonin 60 was reduced to 6.7% and 3.5%, respectively, of the total soluble protein. The N-terminal amino acid sequence of each protein was determined; chaperonin 60 of R. sphaeroides showed 72% identity to E. coli chaperonin 60 protein, and R. sphaeroides chaperonin 10 showed 45% identity with E. coli chaperonin 10. R. sphaeroides chaperonin 60 catalyzed ATP hydrolysis with a specific activity of 134 nmol min-1 mg-1 (kcat = 0.13 s-1) and was inhibited by R. sphaeroides chaperonin 10, but not E. coli chaperonin 10. The E. coli chaperonin 60 ATPase activity was inhibited by chaperonin 10 from both R. sphaeroides and E. coli.  相似文献   

19.
We have identified a mutation of human gamma-interferon (IFN gamma) causing a temperature-sensitive phenotype. We used a randomized oligonucleotide to mutagenize a synthetic human IFN gamma gene, then screened the resulting mutants produced in Escherichia coli for proteins with altered biological activity. One mutant protein selected for detailed characterization exhibited less than 0.3% of the specific biological activity of native IFN gamma in an antiviral activity assay performed at 37 degrees C. However, the protein bound the human IFN gamma receptor with native efficiency at 4 degrees C. Sequencing the plasmid DNA encoding this protein showed that the mutation changed the lysine residue at amino acid 43 to glutamic acid (IFN gamma/K43E). Site-specific mutagenesis at amino acid 43 showed that this protein's phenotype resulted from positioning a negative charge at position 43. Structural characterization of IFN gamma/K43E using CD demonstrated that the protein had native conformation at 25 degrees C, but assumed an altered conformation at 37 degrees C. IFN gamma/K43E in this altered conformation bound poorly to the IFN gamma receptor at 37 degrees C, providing a rationale for the mutant's decreased antiviral activity.  相似文献   

20.
A chaperonin has been purified from a thermophilic bacterium, Thermus thermophilus. It consists of two kinds of proteins with approximate Mr 58,000 and 10,000 and shows a 7-fold rotational symmetry from the top view and a "football"-like shape from the side view under the electron microscopic view. Its weak ATPase activity is inhibited by sulfite and activated by bicarbonate. ATP causes change of its mobility in nondenaturating polyacrylamide gel electrophoresis. The T. thermophilus chaperonin can promote in vitro refolding of several guanidine HCl-denatured enzymes from thermophilic bacteria. At high temperatures above 60 degrees C, where the native enzymes are stable but their spontaneous refoldings upon dilution of guanidine HCl fail, the chaperonin induces productive refolding in an ATP-dependent manner. No or very poor refolding is induced when the chaperonin is added to the solution aged after dilution. An excess amount of the chaperonin is inhibitory for refolding. At middle temperatures (30-50 degrees C), where spontaneous refoldings of the enzymes occur, the chaperonin arrests refolding in the absence of ATP and refolding is induced when ATP is supplemented. At temperatures below 20 degrees C, where spontaneous refoldings also occur, the chaperonin arrests the refolding but ATP does not induce refolding.  相似文献   

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