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J D Doran M Nomizu S Takebe R Ménard D Griffith E Ziomek 《European journal of biochemistry》1999,263(1):145-151
The autocatalytic processing of the streptococcal cysteine protease zymogen (proSCP) to active streptococcal cysteine protease (SCP) was investigated in vitro using purified protein from Streptococcus pyogenes strain B220. It was found that the autocatalytic maturation of the zymogen proceeds through the sequential appearance of at least six intermediates, five of which were characterized through a combination of N-terminal sequencing and MS. Intermediates were identified as resulting from cleavages after Lys26, Asn41, Lys101, Ala112, and Lys118. Time-course studies of the proSCP processing gave a sigmoidal activity profile and indicated that proSCP catalyses its own transformation, mainly via an intermolecular processing mechanism. A similar sequential appearance of intermediates was observed when inactive Cys192Ser proSCP was treated with native, enzymatically active SCP, thus demonstrating that the maturation can exclusively proceed by a bimolecular mechanism. It was shown that proSCP, but not mature SCP, immobilized on a Sepharose resin is capable of liberating itself from the column, indicating that the zymogen is also capable of intramolecular processing. In order to test whether the amino acid sequences at the processing sites could be used for developing new, specific substrates, 3-amino benzoic acid octapeptide derivatives based on all five characterized amino acid sequences from the autoprocessing cleavage sites were synthesized and tested for activity. The 3-amino benzoic acid derivatives have kcat/KM values ranging from 1200 to 7700.M-1.s-1, making them very good endopeptidase substrates for SCP. 相似文献
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What is the role of the transit peptide in thylakoid integration of the light-harvesting chlorophyll a/b protein? 总被引:13,自引:0,他引:13
Whereas it is widely accepted that the transit peptide of the precursor for the light-harvesting chlorophyll a/b protein (preLHCP) is responsible for targeting this polypeptide to chloroplasts, the signals which govern its intraorganellar targeting appears to be transit peptide-mediated for plastocyanin (Smeekins, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375) and several other nuclear-encoded, thylakoid luminal proteins. To determine whether a similar mechanism operates for LHCP (an integral thylakoid protein), we have used oligonucleotide-directed mutagenesis to delete the proposed transit sequence from a petunia precursor of this polypeptide. Intact preLHCP and the deletion mutant product have been expressed in vitro, and their abilities to integrate into purified thylakoids have been compared. We have found that both polypeptides insert into thylakoids correctly, provided the latter are supplemented with a membrane-free stromal extract and Mg.ATP. Our results clearly demonstrate that whereas the transit peptide is required for transport into chloroplasts, thylakoid integration of preLHCP is determined by mature portions of the polypeptide. In addition, we note that transit peptide removal has little effect on the apparent solubility of the in vitro translation products. 相似文献
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High-level expression of the Streptomyces clavuligerus isopenicillin N synthase gene in Escherichia coli. 下载免费PDF全文
The pcbC gene, which encodes isopenicillin N synthase (IPNS), was subcloned from Streptomyces clavuligerus into Escherichia coli by using the pT7 series of plasmid vectors. The polymerase chain reaction was used to introduce an NdeI site at the translation initiation codon of pcbC, allowing the gene to be inserted behind an E. coli type of ribosome binding site. This construction directed high-level expression of IPNS, but the IPNS was in an inactive form in inclusion bodies. Active IPNS was recovered by solubilizing and renaturing the protein. 相似文献
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IS900 PCR to detect Mycobacterium paratuberculosis in retail supplies of whole pasteurized cows' milk in England and Wales. 总被引:6,自引:1,他引:5 下载免费PDF全文
D Millar J Ford J Sanderson S Withey M Tizard T Doran J Hermon-Taylor 《Applied microbiology》1996,62(9):3446-3452
IS900 PCR for Mycobacterium paratuberculosis was applied to cream, whey, and pellet fractions of centrifuged whole cows' milk. The test and simultaneous control reactions gave correct results for spiked milk and for native milk samples obtained directly from M. paratuberculosis-free, subclinically infected, and clinically infected cows. The test was then applied to units of whole pasteurized cows' milk widely obtained from retail outlets throughout central and southern England from September 1991 to March 1993. With peak periods in January to March and in September to November, when up to 25% of units were affected, an overall 22 of 312 samples (7%) tested positive for M. paratuberculosis. In 18 of the 22 positive samples (81%), the PCR signal segregated to the cream or pellet fractions or both, consistent with the presence of intact mycobacteria. Nine of 18 PCR-positive milk samples (50%) and 6 of 36 PCR-negative milk samples (16%) yielded long-term liquid cultures which tested positive for M. paratuberculosis after 13 to 40 months of incubation, despite overgrowth by other organisms. Taken together with data on the prevalence of M. paratuberculosis infection in herds in the United Kingdom, the known secretion of M. paratuberculosis in milk from subclinically infected animals, and the inability of laboratory conditions simulating pasteurization to ensure the killing of all these slowly growing or unculturable organisms, there is a high risk, particularly at peak times, that residual M. paratuberculosis will be present in retail pasteurized cows' milk in England. 相似文献
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Foaming and cell flotation in suspended plant cell cultures and the effect of chemical antifoams 总被引:1,自引:0,他引:1
Foam development and stability in Atropa belladonna suspensions were investigated as a function of culture conditions. Foaming was due mainly to properties of the cell-free broth and was correlated with protein content; effects due to presence of cells increased towards the end of batch culture. Highest foam levels were measured 11 days after inoculation. Air flow rate was of major importance in determining foam volume; foam volume and stability were also strongly dependent on pH. Foam flotation of plant cells was very effective. After 30 min foaming, ca. 55% of cells were found in the foam; this increased to ca. 75% after 90 min. Polypropylene glycol 1025 and 2025, Pluronic PE 6100, and Antifoam-C emulsion were tested as chemical antifoams. Polypropylene glycol 1025 and Antifoam C at concentrations up to 600 ppm had no adverse effect on growth in shake flasks; Pluronic PE 6100 has an inhibitory effect at all levels tested. Concentrations of polypropylene glycol 2025 and Pluronic PE 6100 as low as 20 ppm reduced foam volumes by a factor of ca. 10. Addition of antifoam reduced k(L)a values in bubble-column and stirred-tank bioreactors. After operation of a stirred reactor for 2 days using Antifoam C for foam control, cell production was limited by oxygen due to the effect of antifoam on mass transfer. Theoretical analysis showed that maximum cell concentrations and biomass levels decline with increasing reactors working volume due to greater consumption of antifoam to prevent foam overflow. The results indicate that when chemical foam control is used in plant cell cultures, head-space volume and tolerable foam levels must be considered to optimize biomass production. (c) 1994 John Wiley & Sons, Inc. 相似文献
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The pcbC gene, which encodes isopenicillin N synthase (IPNS), was subcloned from Streptomyces clavuligerus into Escherichia coli by using the pT7 series of plasmid vectors. The polymerase chain reaction was used to introduce an NdeI site at the translation initiation codon of pcbC, allowing the gene to be inserted behind an E. coli type of ribosome binding site. This construction directed high-level expression of IPNS, but the IPNS was in an inactive form in inclusion bodies. Active IPNS was recovered by solubilizing and renaturing the protein. 相似文献
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In 4 years of 24 fetuses at risk for various sphingolipid and mucopolysaccharide storage diseases were examined. Amniocentesis at 16 weeks'' gestation was followed in most cases by culture of amniotic fluid cells and measurement in the cells of the activity of the enzyme suspected to be deficient. Six fetuses were affected; five were examined morphologically and biochemically after abortion. Two fetuses had Tay-Sachs disease, two had GM1 gangliosidosis and one had Hurler''s syndrome. Although in each affected detus the specific enzyme activity was absent, we found in the placenta 5 to 50% of the normal activity. 相似文献