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1.
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
2.
Zi-gang Tian Tian-tang Dong Ya-lin Yang Da Teng Jian-hua Wang 《Applied microbiology and biotechnology》2009,83(1):143-149
The tandem repeats of LFB15(W4,10)-HP(4-16) (LH) gene were cloned into vector pET32a(+) for recombinant expression in Escherichia coli. The E. coli C43(DE3) was successfully used as the expression host to avoid the cell death during induction in E. coli BL21(DE3). Fusion LH dimer was expressed as inclusion body at a portion of 35% of total cell protein and could be well purified
by Ni2+-chelating chromatography. The recombinant LH was released by the cleavage of 50% formic acid, and its yield reached 11.3 mg/l
with purity of 95%. The MIC50 of 3.6 and 1.9 μM of recombinant LH against E. coli CMCC 44102 and Bacillus subtilis ATCC 6633 were determined, respectively. The results demonstrated that expression of tandem LH gene in E. coli C43(DE3) and formic acid cleavage would provide a potent efficient platform for the production of interested peptides.
Zi-gang Tian and Tian-tang Dong contributed equally to this paper. 相似文献
3.
Chang Soo Kang Seung-Yeol Son In Seok Bang 《Journal of microbiology (Seoul, Korea)》2008,46(6):656-661
The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine
at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn
(HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector
to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15–20% of the total cell
proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully
by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography,
and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined
by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial
activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the
amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus. 相似文献
4.
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
5.
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
6.
To clarify the diversity and function of isozymes of ascorbate peroxidase (APX) in plants, a method of producing large quantities of these proteins is needed. Here, we describe an Escherichia coli expression system for the rapid and economic expression of two rice APX genes, APXa and APXb (GeneBank accession Nos. D45423 and AB053297, respectively). The two genes were cloned into the pGEX-6p-3 vector to allow expression of APX as a glutathione-S-transferase (GST) fusion protein. The GST-APXa and GST-APXb fusion proteins were purified by affinity chromatography using a glutathione-Sepharose 4B column, with final yields of 40 and 73 mg g–1 dry cells, respectively. Specific activities were 15 and 20 mM ascorbate min–1 mg–1 protein, respectively. The Km values for ascorbate were 4 and 1 mM, respectively, and those for H2O2 were 0.3 and 0.7 mM, respectively indicating that the two rice isoenzymes have different properties.Revisions requested 27 September 2004; Revisions received 12 November 2004 相似文献
7.
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
8.
A novel production method in Escherichia coli for an antimicrobial peptide of 21 amino acids, buforin IIb, which is a synthetic analog of buforin II, has been developed.
The buforin IIb gene was cloned into the vector pET32a to construct an expression vector pET32a–buforin IIb. The fusion protein
Trx-buforin IIb, purified by nickel nitrilo-triacetic acid (Ni-NTA) resin chromatography, was cleaved by hydroxylamine hydrochloride
to release recombinant buforin IIb. Purification of recombinant buforin IIb was achieved by HPLC: about 3.1 mg/l active recombinant
buforin IIb with purity >99% was obtained. The recombinant buforin IIb showed antimicrobial activities that were similar to
the synthetic one. 相似文献
9.
Chang Soo Kang Chang Wook Park In Seok Bang 《Biotechnology and Bioprocess Engineering》2008,13(3):377-382
The antibacterial peptide hinnavin II, isolated from the cabbage butterfly Artogeia rapae, is synthesized with an amidated lysine 37 residue at C-terminus. Glycine-extended native hinnavin II (hinnavin II-38-Gly,
hin II) gene with 114 bp coding region was cloned in the expression vector pET-32a (+) to construct a fusion expression plasmid
and transformed into Escherichia coli BL21 (DE3) pLysS. The recombinant fusion protein Trx-hin II was expressed in soluble form, purified successfully by Ni2+-chelating chromatography, and cleaved by enterokinase to release recombinant hin II (rhin II). Purification of the rhin II
was achieved by reversed-phase FPLC, and 2.45 mg pure active rhin II was obtained from 800 mL E. coli culture. The molecular mass of the rhin II determined by MALDI-TOF mass spectrometry is consistent with the theoretical molecular
mass of 4,195.0 Da. The purified rhin II showed antimicrobial activities against tested E. coli K 12, E. coli BL21 (DE3), Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus. The application of this expression/purification approach represents a fast and efficient method to prepare milligram quantities
of hinnavin II in its biologically active form. 相似文献
10.
The alyPEEC gene encoding alginate lyase from marine bacterium Pseudoalteromonas elyakovii IAM 14594 was subcloned into pBAD24 with arabinose promoter and sequenced, and overexpressed in TOP10 strain of E. coli after arabinose induction. Expression levels of alyPEEC gene in E. coli cells were over 39.6-fold higher than those in P. elyakovii IAM 14594 cells. The molecular mass of purified alginate lyase from the engineered E. coli cells was estimated to be 32.0 kDa. Optimum pH and temperature of the alginate lyase activity were 7.0 and 30 °C, respectively.
The enzyme was unstable on heating and in acidic and alkaline solution. The enzyme activity was stimulated by the MgCl2, NaCl, KCl, CaCl2, BaCl2 and MnCl2, but was inhibited by the addition of 1.0 mM of EGTA, EDTA, SDS, ZnSO4, AgNO3, and CoCl2. All the alginate, polyM and polyG could be converted into oligosaccharides with more than tetrasaccharides by the purified
recombinant alginate lyase, suggesting that the recombinant alginate lyase produced by the engineered E. coli has highly potential application in seaweed genetics, food and pharmaceutical industries. 相似文献
11.
A pair of bifunctional expression vectors, pBL-WZX and pHY-WZX, for Escherichia coli and Bacillus licheniformis was constructed to express interesting genes in a secretory manner. The vectors contain an expression cassette consisted
of the promoter and signal peptide region of B. licheniformis amyL as well as an artificial multiple cloning site and a terminator and utilize kanamycin-resistance and/or tetracycline-resistance
for selection in both B. licheniformis and E. coli. Both vectors contain a part of 3′ terminal fragment of B. licheniformis amyL. The 5′-terminal or 3′-terminal fragment of B. licheniformis amyL can cause the integration and amplification of expression cassette in the chromosome of B. licheniformis under a kanamycin-selection pressure. pBL-WZX is an integrational vector while pHY-WZX is free one for B. licheniformis. Both vectors were succeeded in secretory expression of manL in both B. licheniformis and E. coli. 相似文献
12.
Adenoregulin is a member of dermaseptin family which are vertebrate antibiotic peptides having lethal effects against a broad spectrum of bacteria, fungi and protozoa. The 99 bp adenoregulin gene was cloned in the expression vector pET32a and transformed into Escherichia coli BL21(DE3). In fed-batch cultivation of BL21(DE3)/pET32a-adr, an exponential feeding strategy was applied to gain 60 g dry cells l–1. The recombinant fusion protein Trx-ADR was expressed in a soluble form. The fusion protein was isolated by Ni2+-chelating chromatography, cleaved with CNBr and purified to homogeneity through reverse phase-HPLC and size exclusion-HPLC. The purified recombinant adenoregulin had antibacterial activity against Escherichia coli K12D31 with apparent Mr of 3.4 kDa, identical to the anticipated value. 相似文献
13.
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
14.
The gene for phospholipase D (PLD) of Streptomyces sp. YU100 was cloned from λ phage library and hetero-logously expressed in Escherichia coli. Using an amplified gene fragment based on the consensus sequences of streptomycetes PLDs, λ phage library of Streptomyces sp. YU100 chromosomal DNA was screened. The sequencing result of BamHI-digested 3.8 kb fragment in a positive phage clone revealed the presence of an open reading frame of a full sequence of
PLD gene encoding a 540-amino acid protein including 33-amino acid signal peptide. The deduced amino acid sequence showed
a high homology with other Streptomyces PLDs, having the highly conserved ‘HKD’ motifs. The PLD gene excluding signal peptide sequence was amplified and subcloned
into a pET-32b(+) expression vector in E. coli BL21(DE3). The recombinant PLD was purified by nickel affinity chromatography and compared the enzyme activity with wild-type
PLD. The results imply that the recombinant PLD produced by E. coli had the nearly same enzyme activity as PLD from Streptomyces sp. YU100. 相似文献
15.
16.
Fábio Cristiano Angonesi Brod Márcia Regina Pelisser Jean Borges Bertoldo Javier Vernal Carlos BlochJr Hernán Terenzi Ana Carolina Maisonnave Arisi 《Molecular biotechnology》2010,44(2):110-119
Staphylococcus xylosus is a microorganism involved in fermentation of meat products and also a natural producer of extracellular lipases. The aim
of the present work was to clone and express in E. coli a lipase from S. xylosus (AF208229). This lipase gene (1084 bp) was amplified from a S. xylosus strain isolated from naturally fermented salami and introduced in pET14b expression vector in order to express the recombinant
fusion protein (histidine-tagged lipase) in E. coli. Recombinant histidine-tagged S. xylosus lipase was purified by affinity chromatography in an HPLC system. The histidine-tagged lipase is a monomer in solution, as
determined by size-exclusion chromatography. It presents a high lipase activity at pH 9.0 and 42°C for p-nitrophenyl acetate and p-nitrophenyl butyrate, among seven different esters assayed (pNPC2, pNPC4, pNPC10, pNPC12, pNPC14, pNPC16, pNPC18). Moreover, the enzyme presented a quite interesting thermal stability, after an incubation period of 10 min at 95°C, 77%
of the initial activity was retained. 相似文献
17.
PEDF34, a functional epitope of pigment epithelium-derived factor (PEDF), obtained by chemical synthesis previously, shows
potential anti-angiogenesis activity described before. We perform a novel method in this study for the expression and purification
of recombinant PEDF34 in E. coli, and make it convenient, soluble and high yield to obtain this small peptide of PEDF. Human PEDF34 gene was cloned into the
fusion-protein expression vector pGEX-4T-1, and the recombinant plasmid was transformed into E. coli strain BL21-DE3. GST-PEDF34 fusion protein was expressed, purified using chromatograph and identified by Western blotting.
The purified fusion protein was digested by thrombin, and the small PEDF34 peptide was isolated by ultrafiltration. Circular
dichroism (CD) analysis identified that secondary structure of PEDF34 mainly characterizes as α-helix. The 34-AA small peptide
could cell-type-specifically inhibit viability of HUVECs in a dose-dependent manner and induce apoptosis of HUVECs. These
results suggested that this type of recombinant PEDF34 may have potential in the treatment of angiogenesis-related diseases
such as solid tumor. 相似文献
18.
Plant defensins are small, highly stable, cysteine-rich antimicrobial peptides produced by the plants for inhibiting a broad-spectrum
of microbial pathogens. Some of the well-characterized plant defensins exhibit potent antifungal activity on certain pathogenic
fungal species only. We characterized a defensin, TvD1 from a weedy leguminous herb, Tephrosia villosa. The open reading frame of the cDNA was 228 bp, which codes for a peptide with 75 amino acids. Expression analyses indicated
that this defensin is expressed constitutively in T. villosa with leaf, stem, root, and seed showing almost similar levels of high expression. The recombinant peptide (rTvD1), expressed
in the Escherichia coli expression system, exhibited potent in vitro antifungal activity against several filamentous soil-borne fungal pathogens.
The purified peptide also showed significant inhibition of root elongation in Arabidopsis seedlings, subsequently affecting the extension of growing root hairs indicating that it has the potential to disturb the
plant growth and development. 相似文献
19.
F. L. Zhang Z. M. Chi K. L. Zhu J. Li M. J. Li L. K. Liang L. F. Wu 《World journal of microbiology & biotechnology》2007,23(3):331-337
The full length empA gene encoding Vibrio anguillarum metalloprotease was amplified by PCR and fused to the expression vector pBAD24. The carboxy-terminal 6xHis-tagged recombinant
metalloprotein (rEmpA) was expressed from plasmid pBAD-VAP6his in E. coli TOP10 and purified with affinity chromatography using a Ni-NTA column. SDS-PAGE analysis and Western blotting revealed a
molecular mass of the mature rEmpA predicted to be 36 kDa. The optimal temperature and pH for the purified rEmpA were 37°C
and 8.0, respectively. The enzyme was stable below 30°C and between pH 5.0 and 8.0, respectively. The results show that Ca2+, Na+ and Mg2+ had an activating effect on the enzyme while Zn2+ and Cu2+ acted as inhibitors of the enzyme. The purified rEmpA was characterized as a zinc metalloprotease as it was inhibited by
zinc- and metal-specific inhibitors, such as 1,10-phenanthroline, EDTA and EGTA. The results indicate that some characteristics
of EmpA from marine V. anguillarum had been modified after expression and processing in the engineered E. coli. The purified rEmpA showed degradation activity towards various kinds of proteins, indicating its potential role in pathogenesis. 相似文献
20.
S-thanatin, a small antimicrobial peptide with 21 amino acid residues, was expressed as a fusion protein containing thrombin
cleavage site in Escherichia coli BL21 (DE3). To reduce the production cost, immobilization of thrombin in polyacrylamide gel for cleavage was studied in this
work. The immobilized thrombin exhibited excellent activity within wider ranges of pH value and temperature for reaction than
free enzyme, and the residual activity could remain above 75% after ten times of usage. Tricine–SDS–PAGE result showed that
the immobilized thrombin could cleave the S-thanatin fusion protein effectively. After cleavage, recombinant S-thanatin was
purified by preparative reversed-phase high-performance liquid chromatography and mass spectrum showed that the molecular
weight (2,448.86) was close to the theoretical value (2,448.98). After purification, about 7 mg of S-thanatin was obtained
from 1 l of culture and the recombinant exhibited excellent bioactivity to E. coli ATCC 25922, with the minimum inhibitory concentration of 12 μg/ml. The purification method could be applied to prepare other
peptides with similar properties at low cost. 相似文献