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1.
Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning,
expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame
(ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant
plasmid was transformed into E. coli
Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis
activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A
gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant
plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed
was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity
to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was
cloned and expressed successfully. It was the basis for further study of Attacin. 相似文献
2.
Design,expression, and characterization of the hybrid antimicrobial peptide T‐catesbeianin‐1 based on FyuA 下载免费PDF全文
The overuse of antibiotics has resulted in the emergence of antibiotic‐resistant bacteria, which presents an urgent need for new antimicrobial agents. At present, antimicrobial peptides have attracted a great deal of attention from researchers. However, antimicrobial peptides often affect a broad range of microorganisms, including the normal flora in a host organism. In the present study, we designed a novel hybrid antimicrobial peptide, expressed the hybrid peptide, and studied its specific target. The hybrid peptide, named T‐catesbeianin‐1, which includes the FyuA‐binding domain of pesticin and the peptide catesbeianin‐1, was designed and expressed in Pichia pastoris X‐33. Then, we determined the antimicrobial activity, cytotoxicity, and specific target of the peptide. T‐catesbeianin‐1 has strong antimicrobial activity and binds to FyuA to inhibit or kill Escherichia coli present in clinical specimens and mixed‐species culture. In summary, these findings suggested that T‐catesbeianin‐1 might be promising and specific antibiotic agent for therapeutic application against fyuA+ E. coli. 相似文献
3.
Wang H Meng XL Xu JP Wang J Wang H Ma CW 《Applied microbiology and biotechnology》2012,94(4):1031-1039
Antimicrobial peptides (AMPs) are widely expressed and play an important role in innate immune defense against infectious
agents such as bacteria, viruses, fungi, and parasites. Cecropins are a family of AMPs synthesized in the fat body of insects
that have proven effective at killing specific pathogens. In order to fulfill their clinical potential as antimicrobial drugs,
a simple, cost-effective method to express AMPs is sorely needed. In this study, we expressed and characterized the cecropin
from Plutella xylostella (pxCECA1) using an intein-dependent expression system in Escherichia coli. We cloned the pxCECA1 gene from larva by RT-PCR and fused the encoding sequence of mature pxCECA1 with an intein gene and
a chitin-binding domain gene (CBD) in pTWIN1 plasmid. The fusion protein CBD–intein–pxCECA1 was expressed in E. coli BL21 (DE3) and separated by flowing cell extracts through a chitin column. Subsequently, self-cleavage of the intein at its
C-terminus was induced in a temperature- and pH-dependent manner, resulting in the release of mature pxCECA1. The optimal
conditions for self-cleavage were determined to be pH 6.0 for 48 h at 4°C, under which 12.3 mg of recombinant pxCECA1 could
be recovered from 1 l of E. coli culture. The purified pxCECA1 displayed antimicrobial activity against a broad variety of gram-positive and gram-negative
bacteria. This preparation was especially effective against Staphylococcus aureus, including methicillin-resistant strains. Catalase release assays demonstrated that pxCECA1 acts as a microbicidal agent.
These results show for the first time that the IMPACT-TWIN expression system is an efficient, cost-effective way to produce
fully functional AMPs and that the AMP pxCECA1 is a novel microbicidal agent with promising therapeutic applications. 相似文献
4.
Youn Hong Jung Ji-Yeun Yi Hyun Jung Jung Yoo Kyung Lee Hong Kum Lee Mahendran Chinnamara Naicker Ji-hyun Uh I Seul Jo Eun Jung Jung Hana Im 《The protein journal》2010,29(2):136-142
A polar bacterium was isolated from Arctic sea sediments and identified as Psychromonas artica, based on 16S rDNA sequence. Psychromonas artica KOPRI 22215 has an optimal growth temperature of 10 °C and a maximum growth temperature of 25 °C, suggesting this bacterium
is a psychrophile. Cold shock proteins (Csps) are induced upon temperature downshift by more than 10 °C. Functional studies
have researched mostly Csps of a mesophilic bacterium Escherichia coli, but not on those of psychrophilic bacteria. In an effort to understand the molecular mechanisms of psychrophilic bacteria
that allow it withstand freezing environments, we cloned a gene encoding a cold shock protein from P. artica KOPRI 22215 (CspAPa) using the conserved sequences in csp genes. The 204 bp-long ORF encoded a protein of 68 amino acids, sharing 56% homology to previously reported E. coli CspA protein. When CspAPa was overexpressed in E. coli, it caused cell growth-retardation and morphological elongation. Interestingly, overexpression of CspAPa drastically increased the host’s cold-resistance by more than ten times, suggesting the protein aids survival in polar environments. 相似文献
5.
Si-Xiu Liu Zhong-Ping Fu Rui-Min Mu Zhi-Bi Hu Fu-Jun Wang Xiang-Rong Wang 《Molecular biology reports》2010,37(4):1781-1786
A nucleic acid sequence MC, encoding Momordica Chanrantia anti-hyperglycaemic peptide MC6 (accession: AAX06814) synthesized according to Escherichia coli preferred codons, was cloned and expressed in E. coli. Recombinant protein pQE8-MC (about 3.5 kDa) was purified and analyzed by 20% SDS–PAGE and western blot. It revealed that
the expressed pQE8-MC had good solubility in aqueous media. An HPLC assay was used to confirm the expression of pQE8-MC. Subsequent
pharmacological activity assay revealed a significant hypoglycemic effect of low dose treatments of pQE8-MC on male kunming
mice. Four hours after an intravenous tail injection, the blood sugar levels of mice treated with pQE8-MC saline solution
A3 (1 mg/kg BW) decreased greatly (P < 0.01) relative to the levels of a control group. This suggests that pQE8-MC, expressed in bioengineered E. coli, has a similar hypoglycemic function to the natural protein MC6 from M. Chanrantia. These results reveal the possibility of using bio-engineered bacteria as an anti-diabetic agent. 相似文献
6.
A peptide antibiotic, gramicidin A, was covalently bound to cystamine self-assembled monolayers on gold surfaces. Each step
of the surface functionalization was characterized by polarization modulation infrared reflection absorption spectroscopy
and X-ray photoelectron spectroscopy. The antimicrobial activity of the anchored gramicidin was tested against three Gram-positive
bacteria (Listeria ivanovii, Enterococcus faecalis, and Staphylococcus aureus), the Gram-negative bacterium Escherichia coli and the yeast Candida albicans. The results revealed that the adsorbed gramicidin reduced, from 60% for E. coli to 90% for C. albicans, the number of culturable microorganisms attached to the surface. The activity was proven to be persistent overtime, up to
6 months after the first use. The bacteria attached to the functionalized surfaces were permeabilized as shown by confocal
microscopy. Taken together, these results indicate a bacteriostatic mode of action of the immobilized peptide. Finally, using
green fluorescent protein-expressing bacteria, it was shown that the development of a bacterial biofilm was delayed on peptide-grafted
surfaces for at least 24 h. 相似文献
7.
Xingjun Feng Chunlong Liu Jiayin Guo Chongpeng Bi Baojing Cheng Zhongyu Li Anshan Shan Zhongqiu Li 《Current microbiology》2010,60(3):179-184
Antimicrobial peptides (AMPs) are extremely attractive candidate for therapeutic agents due to their wide spectrum of antimicrobial
activity and action mechanism different from antibiotics. In this study, a method using genetic engineering for obtaining
an antimicrobial peptide, bovine lactoferricin derivative peptide LfcinB-W10, has been developed. According to the coden usage
of Escherichia coli, a gene encoding the peptide was synthesized and a recombinant vector of E. coli expression pGEX-EN-LFW was constructed. The LfcinB-W10 peptide fused with glutathione S-transferase (GST) was successfully
expressed and about 20 mg fusion protein with 90% purity was obtained from 1 l culture. The recombinant LfcinB-W10 (rLfcinB-W10)
was released from fusion protein by the enterokinase digestion, and about the LfcinB-W10 yield reached 300 μg per 1 l culture.
The purified rLfcinB-W10 was found to have growth inhibition activity against Staphylococcus aureus (S. aureus) ATCC25923. 相似文献
8.
Anna Vacheva Ralitsa Georgieva Svetla Danova Radka Mihova Mariana Marhova Sonia Kostadinova Krasimira Vasileva Maria Bivolarska Stoyanka R. Stoitsova 《Central European Journal of Biology》2012,7(2):219-229
E. coli biofilms cause serious problems in medical practice by contaminating surfaces and indwelling catheters. Due to the rapid
development of antibiotic resistance, alternative approaches to biofilm suppression are needed. This study addresses whether
products released by antagonistic bacteria — Lactobacillus isolates from vaginal and dairy-product samples could be useful for controlling E. coli biofilms. The effects of diluted cell-free supernatants (CFS) from late-exponential Lactobacillus cultures on the growth and biofilm production of Escherichia coli were tested. Most of the CFS applied as 10−2 had no impact on bacterial growth, biofilm development however was influenced even by 10−4 of CFS. Initial screening by crystal violet assay showed that biofilm modulation varied between different CFS and E. coli combinations from inhibition to activation; however three of the tested CFS showed consistency in biofilm suppression. This
was not due to antibacterial activity since Live/Dead fluorescence labeling showed insignificant differences in the amount
of dead cells in control and treated samples. Some E. coli strain-specific mechanisms of response to the three CFS included reduction in hydrophobicity and motility. Released exoploysaccharides
isolated from the three CFS stimulated sessile growth, but proteinase K reduced their inhibitory activities implying participation
of protein or peptide biofilm suppression factor(s). 相似文献
9.
The mercury transporter, merT, from Cupriavidus metallidurans was cloned into pRSET-C and expressed in various E. coli hosts. Expression of merT gene failed in common expression hosts like E. coli BL21(DE3), E. coli BL21(DE3)pLysS and E. coli GJ1158 due to expression induced toxicity. The protein was successfully expressed in E. coli C43(DE3) as inclusion bodies. The inclusion bodies were solubilized with Triton X-100 detergent. The detergent solubilized
protein with N-terminal His-tag was purified in a single-step by immobilized metal affinity chromatography with a yield of 8 mg l−1. 相似文献
10.
11.
The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMARTTM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the
gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide
of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus
sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases,
the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a
(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular
mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of
0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase
had the highest hydrolytic activity towards peanut oil. 相似文献
12.
High level expression and purification of antimicrobial human cathelicidin LL-37 in Escherichia coli
Ján Krahulec Marcela Hyršová Stanislav Pepeliaev Jana Jílková Zbyněk Černý Jana Machálková 《Applied microbiology and biotechnology》2010,88(1):167-175
The human antimicrobial peptide LL-37 is a cationic peptide with antimicrobial activity against both Gram-positive and Gram-negative
microorganisms. This work describes the development of an expression system based on Escherichia coli capable of high production of the recombinant LL-37. The fusion protein Trx-LL-37 was expressed under control of T7 promoter.
The expression of T7 polymerase in the E. coli strain constructed in this work was controlled by regulation mechanisms of the arabinose promoter. The expression plasmid
was stabilized by the presence of parB locus which ensured higher homology of the culture during cultivation without antibiotic selection pressure. This system
was capable of producing up to 1 g of fusion protein per 1 l of culture. The subsequent semipreparative HPLC allowed us to
isolate 40 mg of pure LL-37. LL-37 showed high antimicrobial activity against both Gram-negative and Gram-positive microorganisms.
Its activity against Candida albicans was practically nonexistent. Minimal Inhibition Concentration (MIC) determined for E. coli was 1.65 μM; for Staphylococcus aureus 2.31 μM, and for Enterococcus faecalis 5.54 μM. The effects of cathelicidin on E. coli included the ability to permeabilize both cell membranes, as could be observed by the increase of β-galactosidase activity
in extracellular space in time. Physiological changes were studied by scanning electron microscopy; Gram-positive microorganisms
did not show any visible changes in cell shapes while the changes observed on E. coli cells were evident. The results of this work show that the herein designed expression system is capable of producing adequate
quantities of active human antimicrobial peptide LL-37. 相似文献
13.
K. Srinivasa Babu T. Muthukumaran Aju Antony S. D. Prem Singh Samuel M. Balamurali V. Murugan S. Meenakshisundaram 《Biotechnology letters》2009,31(5):659-664
Human granulocyte-macrophage colony stimulating factor (hGMCSF) is an important therapeutic cytokine. As a novel attempt to
purify hGMCSF protein, without the enzymatic cleavage of the affinity tag, an intein-based system was used. The gene was fused
by overlap extension PCR to the intein sequence at its N-terminal in pTYB11 vector. The hGMCSF was expressed as a fusion protein in E. coli BL21(DE3), and E. coli GJ1158. In the former, the protein was expressed as inclusion bodies and upon purification the yield was 7 mg/l with a specific
activity of 0.5 × 107 IU/mg. In salt-inducible E. coli GJ1158, hGMCSF was expressed in a soluble form at 20 mg/l and a specific activity of 0.9 × 107 IU/mg. The intein-hGMCSF was purified on a chitin affinity column by cleaving intein with 50 mM DTT resulting in a highly
pure 14.7 kDa hGMCSF. 相似文献
14.
Marco Scocchi Christine Lüthy Pietro Decarli Giuseppina Mignogna Philipp Christen Renato Gennaro 《International journal of peptide research and therapeutics》2009,15(2):147-155
Bac7, a cathelicidin peptide of the proline-rich group, inactivates bacteria in a stereospecific manner by entering target
cells without any apparent membrane damage and by binding to as yet unknown intracellular targets. The present study was aimed
at detecting these putative intracellular interactors, which might mediate the antibacterial action of this peptide. By using
affinity resins functionalized with the N-terminal 1-35 fragment of Bac7, a single protein was specifically retained with
high affinity from Escherichia coli cytoplasmic protein lysates. This ligand was identified as the heat shock protein DnaK, the Hsp70 homolog in E. coli. The interaction between the peptide and the chaperone is stereospecific, given that a resin prepared with the all-
d enantiomer failed to retain the protein. In vitro, Bac7(1-35) formed a complex with DnaK with an affinity comparable to that
of other known high-affinity peptide ligands. In addition, at 10–100 μM concentration, the peptide inhibited the protein refolding
activity of the complete DnaK/DnaJ/GrpE/ATP molecular chaperone system in a dose-dependent manner. Despite these results,
the in vitro sensitivity to the peptide, under growth permitting conditions, of DnaK-deficient E. coli strains was not significantly affected compared to the wild-type strain. This suggests that, apart from DnaK, other vital
targets for the proline-rich AMPs are present in susceptible bacteria.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Marco Scocchi and Christine Lüthy contributed equally to this work. 相似文献
15.
Trigger factor (TF) plays a key role as a molecular chaperone with a peptidyl-prolyl cis–trans isomerase (PPIase) activity by which cells promote folding of newly synthesized proteins coming out of ribosomes. Since psychrophilic
bacteria grow at a quite low temperature, between 4 and 15°C, TF from such bacteria was investigated and compared with that
of mesophilic bacteria E. coli in order to offer an explanation of cold-adaptation at a molecular level. Using a combination of gradient PCRs with homologous
primers and LA PCR in vitro cloning technology, the tig gene was fully identified from Psychromonas arctica, whose genome sequence is not yet available. The resulting amino acid sequence of the TF was compared with other homologous
TFs using sequence alignments to search for common domains. In addition, we have developed a protein expression system, by
which TF proteins from P. arctica (PaTF) were produced by IPTG induction upon cloning the tig gene on expression vectors, such as pAED4. We have further examined the role of expressed psychrophilic PaTF on survival
against cold treatment at 4°C. Finally, we have attempted the in vitro biochemical characterization of TF proteins with His-tags
expressed in a pET system, such as the PPIase activity of PaTF protein. Our results demonstrate that the expressed PaTF proteins
helped cells survive against cold environments in vivo and the purified PaTF in vitro display the functional PPIase activity
in a concentration dependent manner. 相似文献
16.
Yaping Lu Qian Lin Jin Wang Yufan Wu Wuyundalai Bao Fengxia Lv Zhaoxin Lu 《Journal of industrial microbiology & biotechnology》2010,37(9):919-925
A Proteus vulgaris strain named T6 which produced lipase (PVL) with nonpositional specificity had been isolated in our laboratory. To produce
the lipase in large quantities, we cloned its gene, which had an opening reading frame of 864 base pairs and encoded a deduced
287-amino-acid protein. The PVL gene was inserted into the Escherichia coli expression vector pET-DsbA, and active lipase was expressed in E. coli BL21 cells. The secretive expression of PVL gene in Bacillus subtilis was examined. Three vectors, i.e., pMM1525 (xylose-inducible), pMMP43 (constitutive vector, derivative of pMM1525), and pHPQ
(sucrose-inducible, constructed based on pHB201), were used to produce lipase in B. subtilis. Recombinant B. subtilis WB800 cells harboring the pHPQ-PVL plasmid could synthesize and secrete the PVL protein in high yield. The lipase activity
reached 356.8 U/mL after induction with sucrose for 72 h in shake-flask culture, representing a 12-fold increase over the
native lipase activity in P. vulgaris. The characteristics of the heterologously expressed lipase were identical to those of the native one. 相似文献
17.
Ruiling Liu Meiqin Liu Jie Liu Yuzhen Chen Yiyin Chen Cunfu Lu 《Plant Growth Regulation》2010,60(2):163-168
A late embryogenesis abundant protein gene, AmLEA from Ammopiptanthus mongolicus, was introduced into Escherichia coli using the IMPACT™-TWIN system to analyze the possible function of AmLEA under heat and cold stresses. A fusion protein about 38 kD was expressed in E.coli cells harboring pTWIN-LEA after the induction of IPTG by SDS–PAGE analysis and the accumulation of the fusion protein peaked
3 h after IPTG addition when cultured at 37°C. Compared with control cells, the E. coli cells expressing AmLEA fusion protein showed improved chilling and heat resistence, illuminating the protein may play a protective
role in cells under stress conditions. These results suggested the natively unstructured protein, similar to other members
of LEA proteins, has high capacity for binding water and potential protective function against dehydration or action similar
to the cold shock chaperones. 相似文献
18.
Wen-Ru Li Xiao-Bao Xie Qing-Shan Shi Hai-Yan Zeng You-Sheng OU-Yang Yi-Ben Chen 《Applied microbiology and biotechnology》2010,85(4):1115-1122
The antibacterial activity and acting mechanism of silver nanoparticles (SNPs) on Escherichia coli ATCC 8739 were investigated in this study by analyzing the growth, permeability, and morphology of the bacterial cells following
treatment with SNPs. The experimental results indicated 10 μg/ml SNPs could completely inhibit the growth of 107 cfu/ml E. coli cells in liquid Mueller–Hinton medium. Meanwhile, SNPs resulted in the leakage of reducing sugars and proteins and induced
the respiratory chain dehydrogenases into inactive state, suggesting that SNPs were able to destroy the permeability of the
bacterial membranes. When the cells of E. coli were exposed to 50 μg/ml SNPs, many pits and gaps were observed in bacterial cells by transmission electron microscopy and
scanning electron microscopy, and the cell membrane was fragmentary, indicating the bacterial cells were damaged severely.
After being exposed to 10 μg/ml SNPs, the membrane vesicles were dissolved and dispersed, and their membrane components became
disorganized and scattered from their original ordered and close arrangement based on TEM observation. In conclusion, the
combined results suggested that SNPs may damage the structure of bacterial cell membrane and depress the activity of some
membranous enzymes, which cause E. coli bacteria to die eventually. 相似文献
19.
Background
Inactive protein inclusion bodies occur commonly in Escherichia coli (E. coli) cells expressing heterologous proteins. Previously several independent groups have found that active protein aggregates or pseudo inclusion bodies can be induced by a fusion partner such as a cellulose binding domain from Clostridium cellulovorans (CBDclos) when expressed in E. coli. More recently we further showed that a short amphipathic helical octadecapeptide 18A (EWLKAFYEKVLEKLKELF) and a short beta structure peptide ELK16 (LELELKLKLELELKLK) have a similar property. 相似文献20.
Bare fiber Bragg grating immunosensor for real‐time detection of Escherichia coli bacteria 下载免费PDF全文
Rajesh Srinivasan Sharath Umesh Swetha Murali Sundarrajan Asokan Sai Siva Gorthi 《Journal of biophotonics》2017,10(2):224-230
Escherichia coli (E. coli) bacteria have been identified to be the cause of variety of health outbreaks resulting from contamination of food and water. Timely and rapid detection of the bacteria is thus crucial to maintain desired quality of food products and water resources. A novel methodology proposed in this paper demonstrates for the first time, the feasibility of employing a bare fiber Bragg grating (bFBG) sensor for detection of E. coli bacteria. The sensor was fabricated in a photo‐sensitive optical fiber (4.2 µm/80 µm). Anti‐E. coli antibody was immobilized on the sensor surface to enable the capture of target cells/bacteria present in the sample solution. Strain induced on the sensor surface as a result of antibody immobilization and subsequent binding of E. coli bacteria resulted in unique wavelength shifts in the respective recording of the reflected Bragg wavelength, which can be exploited for the application of biosensing. Functionalization and antibody binding on to the fiber surface was cross validated by the color development resulting from the reaction of an appropriate substrate solution with the enzyme label conjugated to the anti‐E. coli antibody. Scanning electron microscope image of the fiber, further verified the E. coli cells bound to the antibody immobilized sensor surface.