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1.
The recombinant Bordetella pertussis CyaA pore-forming (CyaA-PF) fragment was previously shown to be expressed separately in Escherichia coli as a soluble precursor that can be in vivo palmitoylated to exert haemolytic activity. In this study, PCR-based mutagenesis
was employed to investigate the contributions to haemolysis of five predicted helices within the N-terminal hydrophobic region
of the CyaA-PF fragment. Single proline substitutions were made for alanine near the centre of each predicted helix as a means
of disrupting local secondary structure. All mutant proteins were over-expressed in E. coli as a 126-kDa soluble protein at levels comparable to the wild-type. Marked reductions in haemolytic activity against sheep
erythrocytes of mutants, A510P, A538P, A583P and A687P pertaining to the putative helices 1500–522, 2529–550, 3571–593 and 5678–698, respectively, were observed. However, a slight decrease in haemolytic activity was found for the proline replacement in
the predicted helix 4602–627 (A616P). MALDI–TOF–MS and LC–MS–MS analyses verified the palmitoylation at Lys983 of all five mutants as identical to that of the CyaA-PF wild-type protein, indicating that toxin modification via this acylation
was not affected by the mutations. Altogether, these results suggest that structural integrity of the predicted helices 1,
2, 3 and 5, but not helix 4, is important for haemolytic activity, particularly for the putative transmembrane helices 2 and
3 that might conceivably be involved in pore formation of the CyaA-PF fragment. 相似文献
2.
A two-parameter statistical model was used to predict the solubility of 96 putative virulence-associated proteins of Flavobacterium psychrophilum (CSF259-93) upon over expression in Escherichia coli. This analysis indicated that 88.5% of the F. psychrophilum proteins would be expressed as insoluble aggregates (inclusion bodies). These solubility predictions were verified experimentally
by colony filtration blot for six different F. psychrophilum proteins. A comprehensive analysis of codon usage identified over a dozen codons that are used frequently in F. psychrophilum, but that are rarely used in E. coli. Expression of F. psychrophilum proteins in E. coli was often associated with production of minor molecular weight products, presumably because of the codon usage bias between
these two organisms. Expression of recombinant protein in the presence of rare tRNA genes resulted in marginal improvements
in the expressed products. Consequently, Vibrio parahaemolyticus was developed as an alternative expression host because its codon usage is similar to F. psychrophilum. A full-length recombinant F. psychrophilum hemolysin was successfully expressed and purified from V. parahaemolyticus in soluble form, whereas this protein was insoluble upon expression in E. coli. We show that V. parahaemolyticus can be used as an alternate heterologous expression system that can remedy challenges associated with expression and production
of F. psychrophilum recombinant proteins. 相似文献
3.
Kateryna Zelena Holger Zorn Manfred Nimtz Ralf Günter Berger 《Archives of microbiology》2009,191(5):397-402
For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein
with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein
with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to
immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on
MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea,
Ca2+, and hemin. 相似文献
4.
Archaeoglobus fulgidus harbors three consecutive and one distantly located gene with similarity to the oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae (KpOadGAB). The water-soluble carboxyltransferase (AfOadA) and the biotin protein (AfOadC) were readily synthesized in Escherichia coli, but the membrane-bound subunits AfOadB and AfOadG were not. AfOadA was affinity purified from inclusion bodies after refolding and AfOadC was affinity purified from the cytosol. Isolated AfOadA catalyzed the carboxyltransfer from [4-14C]-oxaloacetate to the prosthetic biotin group of AfOadC or the corresponding biotin domain of KpOadA. Conversely, the carboxyltransferase domain of KpOadA exhibited catalytic activity not only with its pertinent biotin domain but also with AfOadC. 相似文献
5.
Farzana Ashrafi Neela L. Nonaka M. H. Rahman S. Suzuki 《World journal of microbiology & biotechnology》2009,25(6):1095-1101
The transferability of the tetracycline (TC) resistance gene tet(M) from marine bacteria to human enteric bacteria was examined by a filter-mating method. Vibrio spp., Lactococcus garvieae, Bacillus spp., Lactobacillus sp., and Paenibacillus sp. were used as donors, and Escherichia coli JM109 and Enterococcus faecalis JH2-2 were used as recipients. The combination of Vibrio spp. and E. coli resulted in 5/68 positive transconjugants with a transfer rate of 10−7 to 10−3; however, no transfer was observed with E. faecalis. In case of L. garvieae and E. faecalis, 6/6 positive transconjugants were obtained with a transfer rate of 10−6 to 10−5; however, no transfer was observed with E. coli. The tet(M) gene of Bacillus, Lactobacillus, and Paenibacillus were not transferred to either E. coli or E. faecalis. tet(M) transfer was confirmed in positive E. coli and E. faecalis transconjugants by polymerase chain reaction (PCR) and Southern hybridization. All the donor strains did not harbor plasmids,
while they all harbored transposon Tn916. In the transconjugants, the transposon was not detected by PCR, suggesting the possible transfer of tet(M) from the marine bacterial chromosome to the recipient chromosome. This is the first report to show that tet(M) can be transferred from marine bacteria to human enteric bacteria in a species-specific manner. 相似文献
6.
V. N. Verbenko L. V. Kuznetsova E. P. Krupyan V. I. Shalguev 《Russian Journal of Genetics》2009,45(10):1192-1199
Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed
1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA
+ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein. 相似文献
7.
Two cDNAs isolated from Cymodocea nodosa, CnSOS1A, and CnSOS1B encode proteins with high-sequence similarities to SOS1 plant transporters. CnSOS1A expressed in a yeast Na+-efflux mutant under the control of a constitutive expression promoter mimicked AtSOS1 from Arabidopsis; the wild type cDNA did not improve the growth of the recipient strain in the presence of Na+, but a cDNA mutant that expresses a truncated protein suppressed the defect of the yeast mutant. In similar experiments,
CnSOS1B was not effective. Conditional expression, under the control of an arabinose responsive promoter, of the CnSOS1A and CnSOS1B cDNAs in an Escherichia coli mutant defective in Na+ efflux was toxic, and functional analyses were inconclusive. The same constructs transformed into an E. coli K+-uptake mutant revealed that CnSOS1A was also toxic, but that it slightly suppressed defective growth at low K+. Truncation in the C-terminal hydrophilic tail of CnSOS1A relieved the toxicity and proved that CnSOS1A was an excellent
low-affinity K+ and Rb+ transporter. CnSOS1B mediated a transient, extremely rapid K+ or Rb+ influx. Similar tests with AtSOS1 revealed that it was not toxic and that the whole protein exhibited excellent K+ and Rb+ uptake characteristics in bacteria. 相似文献
8.
Xin Zhou Zhengping Zhang Xiaohe Jia Yifan Wu Lan Luo Zhimin Yin 《World journal of microbiology & biotechnology》2008,24(8):1267-1272
Bacillus subtilis glutamine synthetase (GS) was highly expressed (about 86% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-glnA, which was induced by 0.4 mM IPTG in LB medium, and maximal theanine-forming activity of
the recombinant GS induced in LB is 6.4 U/mg at a series concentration (0–100 mM) of Mn2+ at optimal pH 7.5. In order to get GS with high theanine-forming activity, safety, and low cost for food and pharmaceutics
industry, M9-A (details are described in “Materials and methods”) and 0.1% (w/v) lactose were selected as culture medium and
inducer respectively. Recombinant GS was also highly expressed (84% of total protein) and totally soluble in M9-A and the
specific activity of the recombinant GS is 6.2 U/mg which is approximate to that (6.4 U/mg) induced in LB in the presence
of 10 mM Mn2+ at optimal pH 7.5. The activity is markedly higher activated by Mn2+ than that by other nine bivalent cations. Furthermore, M9-B (5 μM Mn2+ was added into M9-A) was used to culture the recombinant strain and theanine-forming activity of the recombinant GS induced
in M9-B was improved 20% (up to 7.6 U/mg). Finally, theanine production experiment coupled with yeast fermentation system
was carried out in a 1.0 ml reaction system with 0.1 mg crude GS from M9-B or M9-A, and the yield of theanine were 15.3 and
13.1 g/L by paper chromatography and HPLC, respectively. 相似文献
9.
Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain. 相似文献
10.
11.
Yu YJ Wu SC Chan HH Chen YC Chen ZY Yang MT 《Applied microbiology and biotechnology》2008,81(3):523-532
A novel microbial transglutaminase (TGase) from the cultural filtrate of Streptomyces netropsis BCRC 12429 (Sn) was purified. The specific activity of the purified TGase was 18.2 U/mg protein with an estimated molecular
mass of 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The TGase gene of S. netropsis was cloned and an open reading frame of 1,242 bp encoding a protein of 413 amino acids was identified. The Sn TGase was synthesized
as a precursor protein with a preproregion of 82 amino acid residues. The deduced amino acid sequence of the mature S. netropsis TGase shares 78.9–89.6% identities with TGases from Streptomyces spp. A high level of soluble Sn TGase with its N-terminal propeptide fused with thioredoxin was expressed in E. coli. A simple and efficient process was applied to convert the purified recombinant protein into an active enzyme and showed
activity equivalent to the authentic mature TGase.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
12.
A recombinant allophycocyanin (rAPC), used for treatment of tumors, has been expressed in E. coli which was grown in glucose fed-batch culture in a 30 l fermentor. Recombinant allophycocyanin was purified from soluble E. coli cell lysate using hydrophobic interaction chromatography followed by chromatography using amylose affinity column. The purity of product was greater than 98% and yielded an average of 5.5 g kg−1 dry cells. Recombinant allophycocyanin significantly inhibited H22 hepatoma (p ( 0.01) in mice with inhibition rates ranging from 36% to 62% with doses from 6.25 to 50 mg kg−1 d−1. 相似文献
13.
Heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) are molecular chaperones that ensure that the proteins of the cell are properly folded and functional under both normal and stressful conditions. The malaria parasite Plasmodium falciparum is known to overproduce a heat shock protein 70 (PfHsp70) in response to thermal stress; however, the in vivo function of this protein still needs to be explored. Using in vivo complementation assays, we found that PfHsp70 was able to suppress the thermosensitivity of an Escherichia coli dnaK756 strain, but not that of the corresponding deletion strain (dnaK52) or dnaK103 strain, which produces a truncated DnaK. Constructs were generated that encoded the ATPase domain of PfHsp70 fused to the substrate-binding domain (SBD) of E. coli DnaK (referred to as PfK), and the ATPase domain of E. coli DnaK coupled to the SBD of PfHsp70 (KPf). PfK was unable to suppress the thermosensitivity of any of the E. coli strains. In contrast, KPf was able to suppress the thermosensitivity in the E. coli dnaK756 strain. We also identified two key amino acid residues (V401 and Q402) in the linker region between the ATPase domain and SBD that are essential for the in vivo function of PfHsp70. This is the first example of an Hsp70 from a eukaryotic parasite that can suppress thermosensitivity in a prokaryotic system. In addition, our results also suggest that interdomain communication is critical for the function of the PfHsp70 and PfHsp70-DnaK chimeras. We discuss the implications of these data for the mechanism of action of the Hsp70-Hsp40 chaperone machinery. 相似文献
14.
The hemolytic activity of the cell-free culture supernatant of Anabaena variabilis OL S1 was investigated using the hemolysis of rabbit erythrocytes as an assay. The culture medium of A. variabilis started to exhibit hemolytic activity at the late exponential growth phase, and maximized at the stationary phase. The hemolytic
toxin is heat-stable and can be extracted in dichloromethane. The hemolytic activities under different temperature, light
intensity and pH showed a high correlation with the cell densities (r=0.965, 0.951, 0.865, respectively), and the optimum condition is 28~30°C, pH 7.5~8.0, light intensity 120 μmol photons m−2s−1. The addition of 10~20 μg mL−1 chloramphenicol, an inhibitor of protein synthesis, exhibited no marked suppression on the hemolytic activity. The supplement
of 1~20 μg mL−1 glycerol increased the hemolytic activity significantly, suggesting that synthesis of hemolysin was dependent on carbohydrate
and lipid metabolism. The spectrum of erythrocyte sensitivity to the hemolysin indicated that rabbit erythrocytes were more
sensitive to the hemolysin than were rat and human erythrocytes. Goldfish and cat erythrocytes were, however, insensitive
to the hemolytic toxin of A. variabilis. 相似文献
15.
Bacillus
thuringiensis subsp. kurstaki BUPM255 secretes a chitobiosidase Chi255 having an expected molecular weight of 70.665 kDa. When the corresponding gene,
chi255, was expressed in E. coli, the active form, extracted from the periplasmic fraction of E. coli/pBADchi255, was of about 54 kDa, which suggested that Chi255 was excessively degraded by the action of E. coli proteases. Therefore, in vitro progressive C-terminal Chi255 deleted derivatives were constructed in order to study their
stability and their activity in E. coli. Interestingly, when the chitin binding domain (CBD) was deleted from Chi255, an active form (Chi2555Δ5) of expected size
of about 60 kDa was extracted from the E. coli periplasmic fraction, without the observation of any proteolytic degradation. Compared to Chi255, Chi255Δ5 exhibited a higher
chitinase activity on colloidal chitin. Both of the enzymes exhibit activities at broad pH and temperature ranges with maximal
enzyme activities at pH 5 and pH 6 and at temperatures 50°C and 40°C, respectively for Chi255 and Chi255Δ5. Thus, it was concluded
that the C-terminal deletion of Chi255 CBD might be a nice tool for avoiding the excessive chitinase degradation, observed
in the native chitinase, and for improving its activity. 相似文献
16.
Orlita A Sidwa-Gorycka M Malinski E Czerwicka M Kumirska J Golebiowski M Lojkowska E Stepnowski P 《Biotechnology letters》2008,30(3):541-545
Growth of Ruta graveolens shoots was induced when Bacillus sp. cell lysates were added to the culture medium. Elicitation of coumarin by this lysate was also very effective; the concentrations
of isopimpinelin, xanthotoxin and bergapten increased to 610, 2120 and 1460 μg g−1 dry wt, respectively. It also had a significant effect on the production of psoralen and rutamarin (680 and 380 μg g−1 dry wt) and induced the biosynthesis of chalepin, which was not detected in the control sample, up to 47 μg g−1 dry wt With lysates of the Pectobacterium
atrosepticum, their effect on growth was not so significant and had no effect on the induction of coumarin accumulation. But elicitation
with this lysate was much more effective for inducing the production of furoquinolone alkaloids; the concentrations of γ-fagarine,
skimmianine, dictamnine and kokusaginine rose to 99, 680, 172 and 480 μg g−1 dry wt, respectively. 相似文献
17.
Lee JH Moon YH Kim N Kim YM Kang HK Jung JY Abada E Kang SS Kim D 《Biotechnology letters》2008,30(4):749-754
The gene encoding sucrose phosphorylase (742sp) in Leuconostoc mesenteroides NRRL B-742 was cloned and expressed in Escherichia coli. The nucleotide sequence of the transformed 742sp comprised an ORF of 1,458 bp giving a protein with calculated molecular mass of 55.3 kDa. 742SPase contains a C-terminal amino acid sequence that is significantly different from those of other Leu. mesenteroides SPases. The purified 742SPase had a specific activity of 1.8 U/mg with a K
m of 3 mM with sucrose as a substrate; optimum activity was at 37°C and pH 6.7. The purified 742SPase transferred the glucosyl
moiety of sucrose to cytosine monophosphate (CMP).
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
18.
Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic
expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase
and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic
activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant
strain was induced by 0.7 mmol l−1 isopropyl-β-D- thiogalactopyranoside (IPTG) at 20°C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic
of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases. 相似文献
19.
In the Tat protein export pathway of Gram-negative bacteria, TatA and TatB are homologous proteins that carry out distinct
and essential functions in separate sub-complexes. In contrast, Gram-positive Tat systems usually lack TatB and the TatA protein
is bifunctional. We have used a mutagenesis approach to delineate TatA/B-type domains in the bifunctional TatAd protein from
Bacillus subtilis. This involved expression of mutated TatAd variants in Escherichia coli and tests to determine whether the variants could function as TatA or TatB by complementing E. coli
tatA and/or tatB mutants. We show that mutations in the C-terminal half of the transmembrane span and the subsequent FGP ‘hinge’ motif are
critical for TatAd function with its partner TatCd subunit, and the same determinants are required for complementation of
either tatA or tatB mutants in Escherichia coli. This is thus a critical domain in both TatA and TatB proteins. In contrast, substitution of a series of residues at the
N-terminus specifically blocks the ability of TatAd to substitute for E. coli TatB. The results point to the presence of a universally conserved domain in the TatA/B-family, together with a separate
N-terminal domain that is linked to the TatB-type function in Gram-negative bacteria. 相似文献
20.
Fusarium solani degraded methyl tert-butyl ether (MTBE) and other oxygenated compounds from gasoline including tert-butyl alcohol (TBA). The maximum degradation rate of MTBE was 16 mg protein h and 46 mg/g protein h for TBA. The culture
transformed 77% of the total carbon to 14CO2. The estimated yield for MTBE was 0.18 g dry wt/g MTBE. 相似文献