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1.
Pfu DNA polymerase (Pfu) is a DNA polymerase isolated from the hyperthermophilic archaeon Pyrococcus furiosus. With its excellent thermostability and high fidelity, Pfu is well known as one of the enzymes widely used in the polymerase chain reaction. In this study, the recombinant plasmid pLysS His6-tagged Pfu-pET28a was constructed. His-tagged Pfu was expressed in Escherichia coli BL21 (DE3) competent cells and then successfully purified with the ÄKTAprime plus compact one-step purification system by Ni2+ chelating affinity chromatography after optimization of the purification conditions. The authenticity of the purified Pfu was further confirmed by peptide mass fingerprinting. A bio-assay indicated that its activity in the polymerase chain reaction was equivalent to that of commercial Pfu and its isoelectric point was found to be between 6.85 and 7.35. These results will be useful for further studies on Pfu and its wide application in the future. 相似文献
2.
A plasmid carrying the Deinococcus radiodurans recX gene under the control of a lactose promoter decreases the Escherichia coli cell resistance to UV irradiation and γ irradiation and also influences the conjugational recombination process. The D. radiodurans RecX protein functions in the Escherichia coli cells similarly to the E. coli RecX protein. Isolated and purified D. radiodurans RecX and E. coli RecX proteins are able to replace each other interacting with the E. coli RecA and D. radiodurans RecA proteins in vitro. Data obtained demonstrated that regulatory interaction of RecA and RecX proteins preserves a high degree of conservatism despite all the differences in the recombination reparation system between E. coli and D. radiodurans. 相似文献
3.
L. A. Frank V. V. Borisova T. A. Vereshchagina E. V. Fomenko A. G. Anshits I. I. Gitelson 《Applied Biochemistry and Microbiology》2009,45(2):215-220
The results of the development and utilization of an affine magnetic sorbent with Ni2+ ions immobilized on coal ash microspheres are reported. The applicability of the material in the isolation of Histag proteins is demonstrated by examples of the recombinant green fluorescent protein from Clytia gregaria and the Ca2+ regulated photoprotein obelin from Obelia longissima. The specific sorption capacity of the sorbent was 2–7 mg/cm3 for medium-size proteins (20–30 kDa). The particles are suitable for chromatography with the presence of chaotropic agents and EDTA. They are easy to manipulate as isolation of a target protein takes 30–35 min. On the one hand, the elevated affinity of the sorbent to proteins rich in native histidines may result in a high degree of irreversible sorption; on the other hand, it allows isolation of such proteins without the introduction of artificial polyhistidine fragments. 相似文献
4.
Won-Jae Chi Hyerim Kim Heejung Yoo Young Pil Kim Soon-Kwang Hong 《Biotechnology and Bioprocess Engineering》2016,21(2):321-330
New structural designs of antibody fragments have considerable biotechnological and therapeutic potential. In this study, we describe the construction and functional expression of a cetuximab-based antibody fragment (scFv-CH3, minibody) that exhibits activity against human colon cancer. Heterologous expression in Escherichia coli (E. coli) was improved by optimizing the host cells, signal peptides, induction conditions, and culture media. The recombinant minibody was expressed successfully in the periplasm of E. coli BL21(DE3) and purified by immobilized metal affinity chromatography using a Ni2+-NTA resin. The purified minibody showed high binding affinity to cell-surface epidermal growth factor receptor (EGFR) and exhibited inhibition of EGFR-mediated signal transduction in the human colon cancer cell line HT29 in a similar way by the cetuximab. The minibody also showed significant level of anti-cancer ability in the HT29 colorectal cancer xenograft model, which was lower than that by the cetuximab. 相似文献
5.
Ji-Nam Kang Hong-Gyu Kang Hyeon-Jin Sun Yong-Ik Kwon Dae-Hwa Yang Suk-Min Ko Hyo-Yeon Lee 《Plant biotechnology reports》2016,10(3):171-183
Zoysiagrass (Zoysia Japonica Steud.) is used frequently in golf courses and athletic fields. However, Zoysiagrass suffers from large-patch disease caused by Rhizoctonia solani AG2-2 (IV), which results in physical and economic loss. In this study, two full-length chitinase genes encoding pathogen-related proteins were isolated from zoysiagrass. Structural and expression analyses of these genes were carried out. The two isolated chitinases were classified into class Ib (Zjchi1) and class II (Zjchi2). Zjchi1 and, Zjchi2 expression was high in root and stolen and was induced in seedlings by Rhizoctonia solnai AG2-2 (IV) infection. To assess their antifungal activity, the two chitinases were overexpressed in Escherichia coli and purified using Ni2+ and glutathione affinity column chromatography. The purified recombinant chitinases showed broad-spectrum antifungal activity against Rhizoctonia solnai AG2-2 (IV), Rhizoctonia solnai AG-1 (IA), Rhizoctonia cerealis, Botrytis cinerea, Fusarium culmorum, Fusarium graminearum and Trichoderma reesei. 相似文献
6.
Han Bin Pek Pei Yu Lim Chengcheng Liu Dong-Yup Lee Xuezhi Bi Fong Tian Wong Dave Siak-Wei Ow 《Biotechnology letters》2017,39(5):759-765
Objectives
To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis.Results
The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively.Conclusions
Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.7.
Ashraf Mohabati Mobarez Roya Ahmadrajabi Nima Khoramabadi Ali Hatef Salmanian 《World journal of microbiology & biotechnology》2017,33(9):175
We report a new recombinant fusion protein composed of full-length Legionella pneumophila flagellin A and peptidoglycan-associated lipoprotein (PAL), rFLA-PAL, capable of inducing protective immunity against L. pneumophila. The recombinant protein was over expressed in Escherichia coli strain BL21 (DE3) using pET-28a (+) expression vector (pET28a-flaA-pal) and purified by Ni2+ exchange chromatography. Immunological properties of rFLA-PAL were assessed in a mouse model. Female BALB/c mice, immunized with rFLA-PAL, exhibited a rapid increase in serum antibody concentration against each of its protein portions. Furthermore, a strong activation of both innate and adaptive cell-mediated immunity was observed as indicated by antigen-specific splenocyte proliferation, IFN-γ and IL-12 production, and early production of TNF-α in the serum and in splenocyte cultures which were separately assessed against PAL and FLA. BALB/c mice were challenged with a lethal dose of L. pneumophila intravenously. In a 10-days follow-up after intravenous lethal challenge with L. pneumophila, a 100% survival rate was observed for mice immunized with rFLA-PAL, same as for those immunized with a sublethal dose of L. pneumophila. Based on the potent immune responses observed in mice immunized with rFLA-PAL, this recombinant fusion protein could be a potential vaccine candidate against the intracellular pathogen L. pneumophila. 相似文献
8.
THE ribosomal proteins of Escherichia coli have been extensively purified and characterized1,2; but since the structure of ribosomes is not well understood, we havs studied the linear sequence of the proteins on a ribosomal RNA of E. coli Q13. 相似文献
9.
A. R. Kayumov M. I. Bogachev V. A. Manuvera V. N. Lazarev A. V. Sabantsev T. O. Artamonova S. N. Borchsenius I. E. Vishnyakov 《Molecular Biology》2017,51(1):112-121
In both prokaryotes and eukaryotes, the survival at temperatures considerably exceeding the optimum is supported by intense synthesis of the so-called heat shock proteins (HSPs), which act to overcome the adverse effects of heat stress. Among mycoplasmas (class Mollicutes), which have significantly reduced genomes, only some members of the Acholeplasmataceae family possess small HSPs of the α-crystallin type. Overproduction of a recombinant HSP IbpA (Hsp20) from the free-living mycoplasma Acholeplasma laidlawii was shown to increase the resistance of Escherichia coli to short-term heat shock. It has been long assumed that IbpA prevents protein aggregation and precipitation thereby increasing viability of E. coli cells. Several potential target proteins interacting with IbpA under heat stress were identified, including biosynthetic enzymes, enzymes of energy metabolism, and components of the protein synthesis machinery. Statistical analysis of physicochemical properties indicated that IbpA interaction partners significantly differ in molecular weight, charge, and isoelectric point from other members of the E. coli proteome. Upon shortterm exposure to increased temperature, IbpA was found to preferentially interact with high-molecularweight proteins having a pI of about 5.1, significantly lower than the typical values of E. coli proteins. 相似文献
10.
T. R. Shamsutdinov N. P. Balaban A. M. Mardanova Yu. V. Danilova G. N. Rudeskaya M. R. Sharipova 《Russian Journal of Bioorganic Chemistry》2008,34(3):290-293
The recombinant strain of Bacillus subtilis bearing B. intermedius glutamyl endopeptidase gene in multicopy plasmid Δ58.21 secretes the enzyme to the medium at the phase of slowing of growth and the stationary growth phase with accumulation maxima at 24 and 48 h. Enzyme samples were isolated from the culture liquid after 24 and 48 h of culturing of and were purified up to homogeneity by ion exchange chromatography on carboxymethyl cellulose and HPLC on a MonoS column. The molecular weight of the corresponding proteins was 29 kDa. Both preparations had identical structure, but differed in affinity to the specific substrate Z-Glu-pNA. The effects of Ca2+ ions and specific low-molecular and protein inhibitors on the activity of the enzyme corresponding to various growth phases has been studied. 相似文献
11.
Aabha Gupta Santosh Kumar Tiwari Victoria Netrebov Michael L. Chikindas 《Probiotics and antimicrobial proteins》2016,8(3):161-169
Enterocin LD3 was purified using activity-guided multistep chromatography techniques such as cation-exchange and gel-filtration chromatography. The preparation’s purity was tested using reverse-phase ultra-performance liquid chromatography. The specific activity was tested to be 187.5 AU µg?1 with 13-fold purification. Purified enterocin LD3 was heat stable up to 121 °C (at 15 psi pressure) and pH 2–6. The activity was lost in the presence of papain, reduced by proteinase K, pepsin and trypsin, but was unaffected by amylase and lipase, suggesting proteinaceous nature of the compound and no role of carbohydrate and lipid moieties in the activity. MALDI-TOF/MS analysis of purified enterocin LD3 resolved m/z 4114.6, and N-terminal amino acid sequence was found to be H2NQGGQANQ–COOH suggesting a new bacteriocin. Dissipation of membrane potential, loss of internal ATP and bactericidal effect were recorded when indicator strain Micrococcus luteus was treated with enterocin LD3. It inhibited Gram-positive and Gram-negative bacteria including human pathogens such as Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri, Listeria monocytogenes, Escherichia coli O157:H7, E. coli (urogenic, a clinical isolate) and Vibrio sp. These properties of purified enterocin LD3 suggest its applications as a food biopreservative and as an alternative to clinical antibiotics. 相似文献
12.
Prasannan V. Anu Madathiparambil G. Madanan Ananthakrishnan J. Nair Gangaprasad A. Nair Govinda Pillai M. Nair Perumana R. Sudhakaran Padikara K. Satheeshkumar 《Molecular biotechnology》2018,60(4):302-309
Oligopeptidases are enzymes involved in the degradation of short peptides (generally less than 30 amino acids in size) which help pathogens evade the host defence mechanisms. Leptospira is a zoonotic pathogen and causes leptospirosis in mammals. Proteome analysis of Leptospira revealed the presence of oligopeptidase A (OpdA) among other membrane proteins. To study the role of oligopeptidase in leptospirosis, the OpdA of L. interrogans was cloned and expressed in Escherichia coli with a histidine tag (His-tag). The protein showed maximum expression at 37 °C with 0.5 mM of IPTG after 2 h of induction. Recombinant OpdA protein was purified to homogeneity using Ni-affinity chromatography. The purified OpdA showed more than 80% inhibition with a serine protease inhibitor but the activity was reduced to 30% with the cysteine protease inhibitor. The peptidase activity was increased significantly in the presence of Zn2+ at a neutral pH. Inhibitor assay indicate the presence of more than one active sites for peptidase activity as reported with the OpdA of E. coli and Salmonella. Over-expression of OpdA in E. coli BL21 (DE3) did not cause any negative effects on normal cell growth and viability. The role of OpdA as virulence factor in Leptospira and its potential as a therapeutic and diagnostic target in leptospirosis is yet to be identified. 相似文献
13.
Fei Chen Wei Li Liangzhen Jiang Xiang Pu Yun Yang Guolin Zhang Yinggang Luo 《Journal of industrial microbiology & biotechnology》2016,43(9):1281-1292
Geraniol synthase (GES) catalyzes the conversion of geranyl diphosphate (GPP) into geraniol, an acyclic monoterpene alcohol that has been widely used in many industries. Here we report the functional characterization of CaGES from Camptotheca acuminata, a camptothecin-producing plant, and its application in production of geraniol in Escherichia coli. The full-length cDNA of CaGES was obtained from overlap extension PCR amplification. The intact and N-terminus-truncated CaGESs were overexpressed in E. coli and purified to homogeneity. Recombinant CaGES showed the conversion activity from GPP to geraniol. To produce geraniol in E. coli using tCaGES, the biosynthetic precursor GPP should be supplied and transferred to the catalytic pocket of tCaGES. Thus, ispA(S80F), a mutant of farnesyl diphosphate (FPP) synthase, was prepared to produce GPP via the head-to-tail condensation of isoprenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A slight increase of geraniol production was observed in the fermentation broth of the recombinant E. coli harboring tCaGES and ispA(S80F). To enhance the supply of IPP and DMAPP, the encoding genes involved in the whole mevalonic acid biosynthetic pathway were introduced to the E. coli harboring tCaGES and the ispA(S80F) and a significant increase of geraniol yield was observed. The geraniol production was enhanced to 5.85 ± 0.46 mg L?1 when another copy of ispA(S80F) was introduced to the above recombinant strain. The following optimization of medium composition, fermentation time, and addition of metal ions led to the geraniol production of 48.5 ± 0.9 mg L?1. The present study will be helpful to uncover the biosynthetic enigma of camptothecin and tCaGES will be an alternative to selectively produce geraniol in E. coli with other metabolic engineering approaches. 相似文献
14.
Meng-Shiou?Lee You-Cheng?Hseu Guan-Hua?Lai Wen-Te?Chang Hsi-Jien?Chen Chi-Hung?Huang Meng-Shiunn?Lee Min-Ying?Wang Jung-Yie?Kao Bang-Jau?You Wen-?Hsin?Lin Yi-Yang?Lien
Background
Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention.Results
Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay.Conclusions
Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.15.
The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces β-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA 6803) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or β-carotene, respectively. However, expression of this orthologous cruA 6803 gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA6803 was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and β-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA6803 was assayed in vitro with either lycopene or γ-carotene as substrate, β-carotene was synthesized. These data establish that CruA6803 is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed. 相似文献
16.
I. S. Boksha N. V. Lavrova A. V. Grishin A. V. Demidenko A. M. Lyashchuk Z. M. Galushkina R. S. Ovchinnikov A. M. Umyarov L. R. Avetisian M. Iu. Chernukha I. A. Shaginian V. G. Lunin A. S. Karyagina 《Biochemistry. Biokhimii?a》2016,81(5):502-510
Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by SigmaAldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity. 相似文献
17.
Objectives
To improve the stability and sweetness of the sweet-tasting protein, monellin, by using site-directed mutagenesis and a Pichia pastoris expression system with a GAPDH constitutive promoter.Results
Both wild-type and E2 N mutant of single-chain monellin gene were cloned into the PGAPZαA vector and expressed in Pichia pastoris. The majority of the secreted recombinant protein, at 0.15 g/l supernatant, was monellin. This was purified by Sephadex G50 chromatography. The sweetness threshold of wild-type and E2 N were 30 μg/ml and 20 μg/ml, respectively. Compared with the proteins expressed in Escherichia coli, the thermostability of both proteins was improved. The N-terminal sequence is determinative for the sweetness of the proteins expressed in yeast strains.Conclusions
Site-directed mutagenesis, modification of the N-terminus of monellin, and without the need of methanol induction in P. pastoris expression system, indicate the possibility for large-scale production of this sweet-tasting protein.18.
M. Irfan H. I. Guler A. O. Belduz A. A. Shah S. Canakci 《Applied Biochemistry and Microbiology》2016,52(3):277-286
Geobacillus thermodenitrificans AK53 xyl gene encoding xylanase was isolated, cloned and expressed in Escherichia coli. After purifying recombinant xylanase from G. thermodenitrificans AK53 (GthAK53Xyl) to homogeneity by ammonium sulfate precipitation and ion exchange chromatography, biochemical properties of the enzyme were determined. The kinetic studies for GthAK53Xyl showed KM value to be 4.34 mg/mL (for D-xylose) and Vmax value to be 2028.9 μmoles mg–1 min–1. The optimal temperature and pH for enzyme activity were found out to be 70°C and 5.0, respectively. The expressed protein showed the highest sequence similarity with the xylanases of G. thermodenitrificans JK1 (JN209933) and G. thermodenitrificans T-2 (EU599644). Metal cations Mg2+ and Mn2+ were found to be required for the enzyme activity, however, Co2+, Hg2+, Fe2+ and Cu2+ ions caused inhibitor effect on it. GthAK53Xyl had no cellulolytic activity and degraded xylan in an endo-fashion. The action of the enzyme on xylan from oat spelt produced xylobiose and xylopentose. The reported results are suggestive of a xylanase exhibiting desirable kinetics, stability parameters and metal resistance required for the efficient production of xylobiose at industrial scale. 相似文献
19.
Hee Seon Lim Dong-Ho Seo In-Tae Cha Hyunjin Lee Young-Do Nam Myung-Ji Seo 《World journal of microbiology & biotechnology》2018,34(3):44
A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni–NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including Km and Vmax values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme. 相似文献
20.
Alessandra Stefan Natalia Calonghi Fabrizio Schipani Fabrizio Dal Piaz Giorgio Sartor Alejandro Hochkoeppler 《Biotechnology letters》2018,40(9-10):1355-1363