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1.
《Insect Biochemistry》1990,20(7):659-666
Neuroparsin A isolated from the nervous corpora cardiaca of Locusta migratoria has been completely sequenced. It is made up of two very similar polypeptide chains linked by disulfide bridges. It exhibits four microheterogeneous N-termini which are produced naturally by post-translational enzymatic reaction: Asn-Pro-Ile-Ser-Arg accounts for 70% of the N-terminal sequences, Ile-Ser-Arg (20%), Ser-Arg (5%) and Arg (5%). The remainder of the molecule is a homodimer identical to neuroparsin B. The longest neuroparsin A (Mw = 8759 Da) is partially and progressively transformed into neuroparsin B by a stepwise proteolysis which is probably arrested at a putative disulfide bridge involving Cys2 in neuroparsin B. In spite of slight similarities with numerous other hormones, computed comparison with other sequences from the literature revealed only a low level of homology for neuroparsin. 相似文献
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《Insect Biochemistry》1987,17(7):977-983
Neuroparsin A, a sulfur-containing protein synthesized by the medial part of the brain of Locusta and transported to the corpora cardiaca (CC) via the nervi corporis cardiaci I (Girardie et al., 1987), was satisfactorily isolated using electro-elution. A specific immune serum against electro-eluted neuroparsin A was generated. On serial histological sections of the brain treated with the immune serum, only the median neurosecretory cells [stained in blue following the double staining Victoria blue-paraldehyde fuchsin (Al type)] were revealed using peroxidase-antiperoxidase technique. Inactivation of neuroparsin A by antigen-antibody complex formation following injections of immune serum induced green pigmentation, intermediary forms and precocious sexual maturation. These symptoms also follow juvenile hormone (JH) injections. Injections of immune serum antineuroparsin A or the electro-eluted neuroparsin A produced opposite effects on oocyte growth but had no effect on the rate of JH biosynthesis evaluated by radiochemical assay. The neurohormone neuroparsin A could be the median humoral inhibiting factor of the JH system which was previously demonstrated (Girardie, 1966, 1967) in the central area of the pars intercerebralis. 相似文献
4.
The coding region for theEscherichia coli groEL (chaperonin-60) polypeptide was fused downstream of a pea rubisco small subunit transit peptide coding sequence under the control of a tandem 35S CaMV promoter. Transgenic tobacco plants (Nicotiana tabacum cv. Xanthi) containing this modifiedgroEL gene were produced. The modified groEL polypeptide was correctly imported into chloroplasts and accumulated to high or low levels in different plants. The majority of the modified groEL polypeptide was processed correctly to the mature form within the chloroplasts. Approximately 20% of the imported polypeptides retained a portion of the N-terminal transit peptide (TPgroEL). Both groEL and TPgroEL polypeptides assembled into tetradecameric species in the chloroplasts. In plants accumulating high levels of these products, the majority of the plant chaperonin-60 polypeptides in the chloroplast were present in novel hybrid tetradecameric species containing both bacterial and plant chaperonin-60 polypeptides. In plants accumulating low levels of groEL, the predominant species present appeared to be authentic plant cpn6014 and authentic bacterial groEL14. The growth and development of transgenic and control tobacco plants were indistinguishable.Abbreviations cpn60
chaperonin-60
- cpn10
chaperonin-10
- hsp
heat shock protein
- rubisco
ribulose-1,5-bisphosphate carboxylase-oxygenase
- ssu
small subunit
- spp
stromal processing peptidase
- SDS
sodium dodecyl sulfate
- PAGE
polyacrylamide gel electrophoresis 相似文献
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6.
H. Connaris Shauna M. West David W. Hough Michael J. Danson 《Extremophiles : life under extreme conditions》1998,2(2):61-66
The citrate synthase (CS) gene from the hyperthermophilic Archaeon Sulfolobus solfataricus has been cloned and sequenced. The gene encodes a polypeptide of 378 amino acids with a calculated polypeptide molecular
mass of 42 679. High-level expression was achieved in Escherichia coli and the recombinant citrate synthase was purified to homogeneity using a heat step and dye-ligand affinity chromatography.
This procedure yielded approximately 26 mg of pure CS per liter of culture, with a specific activity of 41 U/mg. The enzyme
exhibited a half-life of 8 min at 95°C. A homology-modelled structure of the S. solfataricus CS has been generated using the crystal structure of the enzyme from the thermoacidophilic Archaeon Thermoplasma acidophilum with which it displays 58% sequence identity. The modelled structure is discussed with respect to the thermostability properties
of the enzyme.
Received: August 10, 1997 / Accepted: October 23, 1997 相似文献
7.
Using an antisense control strategy, we isolated an Aspergillus oryzae mutant that produced low levels of carboxypeptidases (CPases). The mutant TFC-1 expressed the antisense RNA of the structural gene of CPase O and showed about 30% of the CPase activity in the parent strain.
Gel filtration analysis indicated that this mutant decreased the CPase activities not only of CPase O but also of CPase O-1
and O-2. This result indicated that the antisense RNA was able to control the expression of the CPase genes as a group. Using
the mutant as a heterologous protein expression host that produced the low levels of CPases, a stable and higher level of
lysozyme expression could be obtained compared with the wild-type. In vitro proteolytic degradation assay also demonstrated that human lysozyme was degraded by purified CPase O.
Received: 16 June 1997 / Received last revision: 29 August 1997 / Accepted: 15 September 1997 相似文献
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Amino acid sequence of locust neuroparsins 总被引:2,自引:0,他引:2
Neuroparsins A and B were isolated from the nervous part of the corpus cardiaca of Locusta migratoria via a two-step purification procedure. Both consist of two polypeptide chains linked by disulfide bridges. The N-terminal sequence of both native neuroparsins was determined: the N-terminal end of neuroparsin B was unique while that of neuroparsin A showed three different sequences. These sequences were that of neuroparsin B and two others having five and two extra N-terminal residues. Neuroparsin B was found as a homodimer and the complete sequence of the monomer, determined from peptide fragments generated by treatment with cyanogen bromide and endoprotease Glu-C, comprises 78 residues. 相似文献
10.
Molecular cloning, expression and characterization of a chitosanase from Microbacterium sp. 总被引:1,自引:0,他引:1
A gene encoding a chitosanase (mschito) was cloned from Microbacterium sp. OU01. The ORF consists of 801 bp which encoded a polypeptide of 266 amino acid residues. The deduced amino acid sequence
shows 98% identity to that of the chitosanase reported in Pseudomonas sp. A-01. In addition, the fusion protein containing MSCHITO was expressed in E. coli and purified using Ni-NTA affinity chromatography. The purified rMSCHITO protein degraded the chitosan (the degree of deacetylation
of 99%) and produced a mixture of chitooligosaccharides. The MSCHITO is thus an endo-chitosanase. 相似文献
11.
Kawamura-Konishi Y Maekawa S Tsuji M Goto H 《Applied microbiology and biotechnology》2011,90(1):227-234
Tyrosinase is expressed as a 67-kDa protein in Pholiota microspora (synonym Pholiota nameko), whereas the same enzyme purified from fruiting bodies of P. microspora is a 42-kDa protein that is cleaved with a C-terminal 25-kDa polypeptide from the 67-kDa protein. To confirm the role of
C-terminal processing in enzyme activity, we expressed a recombinant 67-kDa tyrosinase in Escherichia coli cells. To obtain a soluble protein, the recombinant tyrosinase was expressed as a thioredoxin fusion protein with an enterokinase-cleavable
site. Enterokinase digestion of the fusion protein produced a recombinant 67-kDa tyrosinase that did not have any catalytic
activity. However, chymotrypsin digestion of the fusion protein produced a recombinant 44-kDa tyrosinase that was catalytically
active and had a 25-kDa cleaved C-terminal. Kinetic parameters of the 44-kDa tyrosinase were similar to those of the 42-kDa
tyrosinase purified from the fruiting bodies. These results suggest that tyrosinase is expressed in P. microspora as a latent 67-kDa proenzyme and is converted to the mature active 42-kDa enzyme by proteolytic processing of the C-terminal. 相似文献
12.
D G Yim H H Sato Y H Park Y K Park 《Journal of industrial microbiology & biotechnology》1997,18(6):402-405
During screening for cyclodextrin-forming microorganisms, an alkalophilic Bacillus sp, which produced high activity of cyclodextrin glycosyltransferase, was isolated and identified as Bacillus firmus. The crude enzyme transformed starch to mainly β-and γ-cyclodextrin. The purified enzyme had an optimum pH of 7.5–8.5 and
its optimum temperature was 65°C, which is the highest optimum temperature as compared to other cyclodextrin glycosyltransferases
except that produced by Bacillus amyloliquefaciens.
Received 06 January 1997/ Accepted in revised form 20 March 1997 相似文献
13.
To elucidate the molecular basis of symptom expression in virus-infected plants, the changes in proteins between tobacco, Nicotiana tabacum cv. Ky57, leaves inoculated with cucumber mosaic virus strain Y [CMV(Y)] and strain O [CMV(O)], were compared by 2-dimensional (2-D) gel electrophoresis. The appearance of chlorotic spots in CMV(Y)-inoculated tobacco leaves accompanied an increase of 3 polypeptides and a decrease in 6 polypeptides, as compared with those in the CMV(O)-inoculated tobacco which showed no clear symptoms. The decrease in the amounts of two polypeptides of 22 and 23 kDa was particularly significant: these two polypeptides were compared with a 24 kDa polypeptide, which co-migrated with them in 2-D gel electrophoresis but did not clearly decrease at an early stage of infection, as well as major other proteins of CMV(Y)-inoculated tobacco leaves. However, the 22, 23 and 24 kDa polypeptides showed the same peptide mapping pattern. Furthermore, the 12 amino acid residues at N-termini of the three polypeptides match those of the extrinsic 23 kDa polypeptide of an oxygen-evolving complex from spinach. A comparative analysis of the 22, 23 and 24 kDa polypeptides in N. tabacum and its ancestral parents, N. sylvestris and N. tomentosiformis, revealed that the 22 kDa polypeptide derives from N. sylvestris and the 23 kDa polypeptide from N. tomentosiformis; the 24 kDa polypeptide derives from both ancestral Nicotiana species. The results indicate that the polypeptides whose amounts differentially decrease with the progress of symptom expression in N. tabacum inoculated with CMV(Y) are one component of the oxygen-evolving complex in photosystem II. 相似文献
14.
Feruloyl-CoA:arabinoxylan-trisaccharide O-hydroxycinnamoyl transferase, which catalyzes the transfer of ferulic acid from Fer-CoA to arabinoxylan-trisaccharide in
the formation of feruloyl arabinoxylan-trisaccharide (Fer-AXX), has been found in an ionically bound fraction and a cytosol
fraction of suspension-cultured rice (Oriza sativa L. cv. Nipponbare) cells. Analysis of reaction products by high-performance liquid chromatography showed the formation of
product A, which is one of the transfer products having the same retention time as authentic Fer-AXX. Product A was purified
by reverse-phase chromatographies to characterize its structure. The isolated product A showed the same ultraviolet spectrum
and molecular weight on fast atom bombardment mass spectrometric analysis as those of authentic Fer-AXX. Alakaline saponification
of product A released ferulic acid and oligosaccharide. The released oligosaccharide consisted of arabinose and xylose in
a molar ratio of 1:2. These results support the identity of product A as feruloylated arabinoxylan-trisaccharide and show
the existence of a feruloyltransferase catalyzing the feruloylation of a hemicellulosic fragment.
Received: 14 July 2000 / Accepted: 22 August 2000 相似文献
15.
《Bioscience, biotechnology, and biochemistry》2013,77(12):1985-1990
A gene coding for human nerve growth factor (hNGF) was constructed for expression under control of the trp promoter in E. coli. The plasmid pTRSNGF contained a synthetic hNGF gene fused, in frame, to the region encoding the β-lactamase signal peptide. The plasmid pTRLNGF contained the same coding sequence as hNGF attached downstream from the N-terminal fragment of the trp L gene. E. coli cells harboring pTRSNGF produced an amount of hNGF constituting 4% of the total cellular protein, and removed the β-lactamase signal peptide. The mature protein hNGF was biologically active in the PC12h bioassay for neurite outgrowth. This biological activity was comparable to that of authentic mouse NGF. E. coli cells harboring pTRLNGF produced an amount of fusion protein hNGF constituting 25% of the total cellular protein. Although the fusion protein hNGF formed inclusion bodies in cells, dissolved fusion protein hNGF was active in neurite outgrowth from PC12h cells. 相似文献
16.
A. Schäfer M. A. Stein K.-H. Schneider F. Giffhorn 《Applied microbiology and biotechnology》1997,48(1):47-52
By polymerase chain reaction mutagenesis techniques, an NdeI restriction site was introduced at the initiation codon of the mannitol dehydrogenase (MDH) gene (mtlK) of Rhodobacter sphaeroides Si4. The mtlK gene was then subcloned from plasmid pAK74 into the NdeI site of the overexpression vector pET24a+ to give plasmid pASFG1. Plasmid pASFG1 was introduced into Escherichia coli BL21(DE3), which was grown in a 1.5-l bioreactor at 37 °C and pH 7.0. Overexpression of MDH in Escherichia coli BL21(DE3) [pASFG1] was determined by enzymatic analysis and sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis.
Under standard growth conditions, E. coli produced considerable amounts of a polypeptide that correlated with MDH in SDS gels, but the activity yield was low. Decreasing
the growth temperature to 27 °C and omitting pH regulation resulted in a significant increase in the formation of soluble
and enzymatically active MDH up to a specific activity of 12.4 U/mg protein and a yield of 26 000 U/l, which corresponds to
0.38 g/l MDH. This was an 87-fold overexpression of MDH compared to that of the natural host R. sphaeroides Si4, and a 236-fold improvement of the volumetric yield. MDH was purified from E. coli BL21(DE3) [pASFG1] with 67% recovery, using ammo-nium sulfate precipitation, hydrophobic interaction chromatography, and
gel filtration. Partial characterization of the recombinant MDH revealed no significant differences to the wild-type enzyme.
Received: 18 February 1997 / Received revision: 27 March 1997 / Accepted: 27 March 1997 相似文献
17.
Human thymus poly(A) polymerase (EC 2.7.7.19) activity has been investigated using poly(A) and oligo(A) as initiators. All obtained fractions reveal more than one polypeptide as detected by immunoblotting after SDS-PAGE. In addition to the homogeneously purified (Tsiapalis et al., J Biol Chem 250: 4486–1496, 1975 and Wahle, J Biol Chem 266: 3131–3139, 1991), about 60 kDa polypeptide, a larger polypeptide, about 80 kDa, that comigrates in the region of poly(A) polymerase activity was detected, enriched and partially characterized; it appears having similar size with bovine poly(A) polymerase cloned in E. coli. Polyclonal antiserum produced against recombinant bovine poly(A) polymerase reacts more efficiently with the about 80 kDa polypeptide upon immunoblotting, and can precipitate the poly(A) polymerase activity. This enzyme form, from human tissue, is novel in terms of size and may reflect intact or physiological form of poly(A) polymerase in human thymus, and supports and substantiates recent reports on the enzyme from other sources. 相似文献
18.
Yasuo Kimura Akinori Suzuki Tetsuo Takematsu Makoto Konnai 《Bioscience, biotechnology, and biochemistry》2013,77(4):1071-1073
The purified polyethylene glycol (PEG) dehydrogenase from cells of a synergistic mixed culture of Flavobacterium and Pseudomonas species showed a similar absorption spectrum to those of other quinoproteins reported so far. The prosthetic group of the PEG dehydrogenase after extraction with cold methanol and purification by DEAE-Sephadex A-25 column chromatography and Sephadex G-25 gel filtration showed the same elution profiles as those of authentic pyrrolo-quinoline quinone (PQQ). Absorption and fluorescence spectra of the purified prosthetic group and its prosthetic group capability for glucose dehydrogenase indicated that it was identical with authentic PQQ.The enzyme was induced during bacterial cell growth on a medium containing PEG 6000 as a sole source of carbon. The purified enzyme oxidized primary alcohols of C2-C16 and the corresponding aldehydes of C4-C7. The enzyme also reacted with nonionic surfactants containing PEG residues. The enzyme reduced 2,6-dichlorophenolindophenol (DCIP) and the Km value for DCIP was calculated to be 1.4 × 10?4m. The DCIP reductase activity was inhibited by carbonyl reagents like semicarbazide, hydrazine, hydroxylamine and 1,4-benzoquinone. 1,4-Benzoquinone inhibited the DCIP reductase activity competitively as to DCIP. 相似文献
19.
A hemolysin produced by Treponema hyodysenteriae ATCC27164 was purified from broth filtrates by acetic and (NH4)2SO4 precipitations followed by ion exchange chromatography on diethylaminoethyl-Sephacel and gel filtration using Ultrogel AcA44. The purified hemolysin displayed only one band on polyacrylamide gel electrophoresis. By gel filtration the molecular weight was estimated as 74,000 daltons. The isolated hemolysin was oxygen resistant, heat labile and was not inactivated over a wide range of pH values. Further analysis indicated that this hemolysin was probably a polypeptide or a protein associated with lipids and nucleotides. Its action on rabbit erythrocytes which did not require any divalent cations could not be related to a lipolytic or proteolytic activity. 相似文献
20.
BLIS 213, is a bacteriocin-like inhibitory substance produced by Carnobacterium piscicola 213. It is active against Carnobacterium, Enterococcus and Listeria spp. No activity was observed against tested Lactobacillus, Lactococcus, Leuconostoc and Pediococcus strains, nor against Gram-negative bacteria. The BLIS 213 activity was inactivated by several proteolytic enzymes. It was
heat resistant (121°C for 20 min), and stable over a pH range of 2–8. Activity was determined by a dilution micromethod;
it was increased after SDS treatment. A mutant strain which lacks bacteriocin production was isolated and designated as
Carnobacterium piscicola 213a. It had the same phenotypic and biochemical properties as the parent strain, and was not sensitive to bacteriocin activity.
The apparent molecular weight of the bacteriocin in the crude extract was greater than 10 kDa. It was about 6 kDa after
SDS-PAGE of a partially purified bacteriocin by adsorption on producer cells. The isoelectric point of the BLIS 213 was around
9.3.
Received 21 January 1997/ Accepted in revised form 25 April 1997 相似文献