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1.
Expression of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fusion partners 总被引:2,自引:0,他引:2
Chen YQ Zhang SQ Li BC Qiu W Jiao B Zhang J Diao ZY 《Protein expression and purification》2008,57(2):303-311
It has been reported that it is difficult to express cationic antibacterial peptides in engineered bacteria because such peptides are highly toxic to the host bacteria cells and sensitive to intracellular proteases. Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi and tumor cells, which may possibly be used as an antimicrobial agent. Here we tried to express ABP-CM4 in Escherichia coli cells using either the GST fusion system or the intein-mediated fusion expression system. In order to investigate the possible use of these two fusion partners in cationic small peptide expression and purification, a mutant ABP-CMt, which is a highly positively charged peptide with +9 charges at neutral pH, was designed. In the present study, we have shown that both ABP-CM4 and ABP-CMt peptides can be expressed and purified by the intein-mediated expression system but not by the GST fusion expression system. Thus the intein-mediated peptide expression and purification system potentially could be employed for the production of recombinant protease-sensitive and cytotoxic peptides. 相似文献
2.
Yu-Chuan Juang Sunil S. Adav Duu-Jong Lee Juin-Yih Lai 《Applied microbiology and biotechnology》2009,85(2):383-388
Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and
tumor cells, which may possibly be used as an antimicrobial agent. We report here the application of small ubiquitin-related
modifier (SUMO) fusion technology to the expression and purification of cationic antibacterial peptide ABP-CM4. The fusion
protein expressed in a soluble form was purified to a purity of 90% by Ni-IDA chromatography and 112 mg protein of interest
was obtained per liter of fermentation culture. After the SUMO–CM4 fusion protein was cleaved by the SUMO protease at 30 °C
for 1 h, the cleaved sample was re-applied to a Ni-IDA. Finally, about 24 mg recombinant CM4 was obtained from 1 l fermentation
culture with no less than 96% purity and the recombinant CM4 had similar antimicrobial properties to the synthetic CM4. Thus,
the SUMO-mediated peptide expression and purification system potentially could be employed for the production of recombinant
cytotoxic peptides. 相似文献
3.
Jian Feng Li Jie Zhang Zhen Zhang Chun Tao Kang Shuang Quan Zhang 《Current microbiology》2011,62(1):296-300
Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and
tumor cells, which may possibly be used as an antimicrobial agent. To improve the expression level of CM4 in Escherichia coli, two tandem repeats of CM4 genes were cloned into the vector pSUMO to construct an expression vector pSUMO–2CM4. The fusion
protein SUMO–2CM4, purified by Ni2+-chelating chromatography, was cleaved by hydroxylamine hydrochloride to release recombinant CM4. After the cleaved sample
was re-applied to a Ni-IDA column, finally, about 48 mg recombinant CM4 was obtained from 1 L bacterial culture with no less
than 96% purity, which was the highest yield of CM4 reported so far. 相似文献
4.
The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic α-helical peptide that exhibits a broad range of antimicrobial activity. To explore
a new approach for the expression of ABP-CM4 in E. coli, the gene ABP-CM4, obtained by recursive PCR (rPCR), was cloned into the vector pET32a to construct a fusion expression plasmid.
The fusion protein Trx-CM4 was expressed in soluble form, purified by Ni2+-chelating chromatography, and cleaved by formic acid to release recombinant CM4. Purification of rCM4 was achieved by affinity
chromatography and reverse-phase HPLC. The purified of recombinant peptide showed antimicrobial activities against E. coli K12D31, Penicillium chrysogenum, Aspergillus niger and Gibberella saubinetii. According to the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html), 116 peptides contain a Met residue, but
only 5 peptides contain the AspPro site, indicating a broader application of formic acid than CNBr in cleaving fusion protein.
The successful application to the expression of the ABP-CM4 indicates that the system is a low-cost, efficient way of producting
milligram quantities of ABP-CM4 that is biologically active. 相似文献
5.
Antifungal mechanism of antibacterial peptide, ABP-CM4, from Bombyx mori against Aspergillus niger 总被引:1,自引:0,他引:1
Antibacterial peptide, CM4 (ABP-CM4), a 35 amino acid peptide from Chinese silkworm—Bombyx mori, displayed a strong antifungal activity against Aspergillus niger, Trichoderma viride and Gibberella saubinetii. Scanning electron microcopy showed that the morphology of conidia became more irregular and swelled when treated with ABP-CM4
at its minimal inhibitory concentration (MIC) of 8 μM. A cell wall regeneration assay indicated that the plasma membrane was
the prime target of ABP-CM4 action. Confocal laser scanning microscopy showed that the cytoskeleton of A. niger was destroyed when treated with ABP-CM4 at 8 μM. Furthermore, transmission electron microscopy showed that the membrane
and the cellular organelles of fungus were disrupted and there were many vacuoles in the fungal cellular space after the treatment
with ABP-CM4. A gel-retardation assay showed that ABP-CM4 bound the DNA of A. niger. Our results suggest that ABP-CM4 exerts its antifungal activity by disrupting the structure of cell membranes and the cytoskeleton
and interacts with the organelles, such as the mitochondrion and with the DNA in the fungal cell, subsequently resulting in
cell death. 相似文献
6.
新型抗菌肽研究有助于解决细菌对抗生素的耐药性问题。本研究用SMART技术构建了景东湍蛙Amolops jingdongensis皮肤的全长cDNA文库。通过单克隆和测序获得一个抗菌肽cDNA序列,序列比对结果表明其属于jindongenin-1家族,命名为jindongenin-1d。其cDNA序列全长321bp,编码含66个氨基酸残基的多肽。该多肽包括1个信号肽和1个前肽序列。成熟jindongenin-1d多肽包含24个氨基酸残基,理论分子量为2 709.38,等电点为9.24。对人工合成的jindongenin-1d蛋白进行了抗菌和溶血活性分析,结果表明jindongenin-1d对所选的革兰氏阴性菌、革兰氏阳性菌和真菌均有显著抑制作用,同时有弱溶血活性。本研究结果有助于进一步了解两栖动物皮肤分泌物活性物质的多态性和新型抗感染药物的设计。 相似文献
7.
8.
Wade D Flock JI Edlund C Löfving-Arvholm I Sällberg M Bergman T Silveira A Unson C Rollins-Smith L Silberring J Richardson M Kuusela P Lankinen H 《Protein and peptide letters》2002,9(6):533-543
Temporin A, 18 analogs, and a cecropin A-temporin A hybrid peptide were tested with antibiotic sensitive and resistant bacteria, fungi, human erythrocytes, and in clotting assays. Several peptides were active in these assays, and some analogs (D-TA, W1-TA, and Con-L4,G10) may be useful lead compounds for further antibiotics development. The activity of temporin A was found to be dependent upon several of its structural features, including amino acid composition and sequence, chirality, helicity, and positive charge. 相似文献
9.
Mina Memarpoor-Yazdi Hadi Zare-Zardini Ahmad Asoodeh 《International journal of peptide research and therapeutics》2013,19(2):99-108
Much research has been focused on antimicrobial peptides (AMPs) derived from insect immune defense reactions due to their potential in the development of new antibiotics. In this study, a new AMP from the insect Paederus dermatitis, named sarcotoxin Pd was identified and purified using gel filtration and reverse-phase high-performance liquid chromatography. Our results showed that this peptide has broad-spectrum inhibitory effects on examined microbes. Sarcotoxin Pd is composed of 34 amino acids and its molecular weight was estimated to be 3613.26 ± 0.5 Da. Minimum inhibitory concentration (MIC) values of sarcotoxin Pd against Gram-negative bacteria were lower than Gram-positive bacteria and fungi. The identified peptide showed the highest antimicrobial effect against Klebsiella pneumonia and Escherichia coli. This peptide did not reveal significant hemolytic activity against human red blood cells particularly in the range of MIC values. Confirming the potential antimicrobial activities of synthetic peptide, this paper addresses the role of sarcotoxin Pd in the treatment of systemic microbial illnesses. 相似文献
10.
抗菌肽的作用机制、生物活性及应用研究进展 总被引:1,自引:0,他引:1
抗菌肽广泛存在于生物界,是辅助生物机体抵抗外来病原体入侵的重要防御分子。抗菌肽不仅能抑制、杀灭多种细菌,而且具有抗真菌、抗寄生虫、抗病毒、抗肿瘤和免疫调节等生物学活性。抗菌肽的作用机制与传统抗生素不同,不仅具有广谱抗微生物作用,而且不易诱导机体产生耐药性,因此,在治疗临床耐药菌株方面具有极大的开发潜力。 相似文献
11.
LS Tavares JV Rettore RM Freitas WF Porto AP Duque Jde L Singulani ON Silva Mde L Detoni EG Vasconcelos SC Dias OL Franco Mde O Santos 《Peptides》2012,37(2):294-300
Antimicrobial peptides (AMPs) are compounds that act in a wide range of physiological defensive mechanisms developed to counteract bacteria, fungi, parasites and viruses. These molecules have become increasingly important as a consequence of remarkable microorganism resistance to common antibiotics. This report shows Escherichia coli expressing the recombinant antimicrobial peptide Pg-AMP1 previously isolated from Psidium guajava seeds. The deduced Pg-AMP1 open reading frame consists in a 168bp long plus methionine also containing a His6 tag, encoding a predicted 62 amino acid residue peptide with related molecular mass calculated to be 6.98kDa as a monomer and 13.96kDa at the dimer form. The recombinant Pg-AMP1 peptide showed inhibitory activity against multiple Gram-negative (E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Staphylococcus epidermides) bacteria. Moreover, theoretical structure analyses were performed in order to understand the functional differences between natural and recombinant Pg-AMP1 forms. Data here reported suggest that Pg-AMP1 is a promising peptide to be used as a biotechnological tool for control of human infectious diseases. 相似文献
12.
Nádia Skorupa Parachin Kelly Cristina MulderAntônio Américo Barbosa Viana Simoni Campos DiasOctávio Luiz Franco 《Peptides》2012
Antimicrobial peptides (AMPs) consist of molecules that act on the defense systems of numerous organisms toward multiple pathogens such as bacteria, fungi, parasites and viruses. These compounds have become extremely significant due to the increasing resistance of microorganisms to common antibiotics. However, the low quantity of peptides obtained from direct purification is, to date, still a remarkable bottleneck for scientific and industrial research development. Therefore, this review describes the main heterologous systems currently used for AMP production, including bacteria, fungi and plants, and also the related strategies for reaching greater functional peptide production. The main difficulties of each system are also described in order to provide some directions for AMP production. In summary, data revised here indicate that large-scale production of AMPs can be obtained using biotechnological tools, and the products may be applied in the pharmaceutical industry as well as in agribusiness. 相似文献
13.
P18 (KWKLFKKIPKFLHLAKKF-NH(2)), an a-helical antimicrobial peptide designed from cecropin Amagainin 2 hybrid, was known to have potent antimicrobial activity against bacteria as well as fungi without hemolytic activity. To find the peptides comparable or superior to the antimicrobial activity of P18, the two reversed peptides (Rev-1 and Rev-2) of P18 were designed and synthesized. These peptides were found to have similar antimicrobial activity against bacterial and fungal cells without hemolytic activity as compared with P18. Furthermore, a reversed peptide, Rev-2 was shown to have a two-fold higher activity in killing some bacterial cells than P18. Therefore, these results suggested that Rev-2 peptide seems to be an excellent candidate for developing novel peptide antibiotics. 相似文献
14.
Human beta-defensins are 2–5 kDa, cationic, microbicidal peptides, which represent the first-line host defense against several
Gram-negative and Gram-positive bacteria, fungi and viruses. They contain a conserved disulfide-bridge pattern of three pairs
of intramolecular cystine bonds. The well-known public health problem related with the growing number of multiresistant bacteria
has driven research to look for novel antibiotics, such beta-defensins and a feasible way to produce them. Heterologous expression
of beta-defensins could be one way to generate large quantities of beta-defensins for clinical research; however, heterologous
expression of beta-defensins has some biochemical problems, such toxicity toward the host cell, peptide degradation by proteolytic
cell enzymes, size, folding constrains and low recombinant peptide yields. In this communication, several heterologous systems
for producing human beta-defensins are reviewed. 相似文献
15.
植物多肽抗生素研究进展 总被引:7,自引:0,他引:7
植物多肽抗生素是一类对细菌、真菌等微生物及某些昆虫和动植物细胞具有抑制或杀灭作用的小分子多肽. 根据多肽抗生素的氨基酸序列及二级结构,可将植物多肽抗生素分为9类,包括硫素(thionins)、植物防御素(plant defensins)、转脂蛋白(lipid transfer proteins, LTPs)、橡胶素(heveins)、打结素(knottins)、凤仙花素(1b-AMPs)和新近发现的荠菜素(shepherdins)、蜕皮素(snakins)、环肽(cyclotides). 对近年来植物多肽抗生素的分类、抗菌机理、生物活性及基因工程等方面的研究情况作一介绍,希望有助于我国在这一领域的研究与开发. 相似文献
16.
A new antibiotic, U-21,963, is produced by a new strain of Trichoderma viride. Antibiotic activity can be demonstrated against both gram-positive and gram-negative bacteria and also against a wide variety of fungi. U-21,963 is not cross-resistant with other commonly used antibiotics. U-21,963 afforded no protection against Klebsiella pneumoniae, Streptococcus pyogenes, or Staphylococcus aureus when it was injected subcutaneously into mice. 相似文献
17.
Y T Liu T I Ho A R Lee C F Chen H Y Chen C J Chen 《Proceedings of the National Science Council, Republic of China. Part B, Life sciences》1986,10(2):98-104
Chounghwamycin A, a new antitumor antibiotic produced by a strain of Streptomyces sp. No. PL-D-5, was isolated and characterized. It appeared to belong to the actinomycin group of antibiotics from physico-chemical studies and has an empirical formula of C63H88N11O21. The antibiotic is extractable into an organic solvent from the fermentation broth, possessing potent antileukemic activity against P388 mouse leukemia in mice and antimicrobial activity against Gram-positive bacteria with MIC values about 0.1-0.4 microgram/ml, but showed no activity on Gram-negative bacteria, yeast and fungi tested. 相似文献
18.
Felicitas Fazius Christoph Zaehle Matthias Brock 《Applied microbiology and biotechnology》2013,97(9):3763-3772
Plants as well as pro- and eukaryotic microorganisms are able to synthesise lysine via de novo synthesis. While plants and bacteria, with some exceptions, rely on variations of the meso-diaminopimelate pathway for lysine biosynthesis, fungi exclusively use the α-aminoadipate pathway. Although bacteria and fungi are, in principle, both suitable as lysine producers, current industrial fermentations rely on the use of bacteria. In contrast, fungi are important producers of β-lactam antibiotics such as penicillins or cephalosporins. The synthesis of these antibiotics strictly depends on α-aminoadipate deriving from lysine biosynthesis. Interestingly, despite the resulting industrial importance of the fungal α-aminoadipate pathway, biochemical reactions leading to α-aminoadipate formation have only been studied on a limited number of fungal species. In this respect, just recently an essential isomerisation reaction required for the formation of α-aminoadipate has been elucidated in detail. This review summarises biochemical pathways leading to lysine production, discusses the suitability of interrupting lysine biosynthesis as target for new antibacterial and antifungal compounds and emphasises on biochemical reactions involved in the formation of α-aminoadipate in fungi as an essential intermediate for both, lysine and β-lactam antibiotics production. 相似文献
19.
20.
Streptomyces bacteria are ubiquitous in soil, conferring the characteristic earthy smell, and they have an important ecological role in the turnover of organic material. More recently, a new picture has begun to emerge in which streptomycetes are not in all cases simply free-living soil bacteria but have also evolved to live in symbiosis with plants, fungi and animals. Furthermore, much of the chemical diversity of secondary metabolites produced by Streptomyces species has most likely evolved as a direct result of their interactions with other organisms. Here we review what is currently known about the role of streptomycetes as symbionts with fungi, plants and animals. These interactions can be parasitic, as is the case for scab-causing streptomycetes, which infect plants, and the Streptomyces species Streptomyces somaliensis and Streptomyces sudanensis that infect humans. However, in most cases they are beneficial and growth promoting, as is the case with many insects, plants and marine animals that use streptomycete-produced antibiotics to protect themselves against infection. This is an exciting and newly emerging field of research that will become increasingly important as the search for new antibiotics switches to unusual and under-explored environments. 相似文献