共查询到20条相似文献,搜索用时 31 毫秒
1.
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
2.
Hydrophobins secreted by filamentous fungi self-assemble into an amphipathic film at hydrophilic/hydrophobic interfaces. This
unique property suggests that the hydrophobins have a high potential for industrial applications. However, the assemblages
of class I hydrophobins are highly insoluble, making such commercial applications difficult. To enhance the solubility of
class I hydrophobins, we have attempted to express class I hydrophobin PNH1 from Pholiota nameko fused with glutathione S-transferase (GST) in Escherichia coli. The GST–PNH1 was effectively isolated from the soluble fraction of transformed E. coli, and subsequent analysis revealed that the purified GST–PNH1 had almost the same emulsifying activity as PNH1. 相似文献
3.
Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of
enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality.
Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers
such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory
strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular
production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies
have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached.
This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that
significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials
and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy. 相似文献
4.
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
5.
Among the numerous bacterial Type II restriction enzymes, EcoRI endonuclease is the most extensively studied and is widely used in recombinant DNA technology. Its heterologous overexpression
as recombinant protein has already been studied. However, very limited information concerning its fused product is available
thus far. In the present study, the EcoRI restriction endonuclease gene was cloned and expressed as a part of maltose-binding fusion protein under the control of
strong inducible tac promoter in TB1 strain of Escherichia coli cells. Transformed cells containing pMALc2X-EcoRI recombinant plasmid were unable to grow under experimental conditions. However, fused EcoRI protein was purified (with the yield of 0.01 mg/l of bacterial culture) by affinity chromatography from E. coli cells induced at the late exponential phase of growth. Restriction quality test revealed that the purified product could
restrict a control plasmid DNA in vitro. 相似文献
6.
Pedersen MH Borodina I Moresco JL Svendsen WE Frisvad JC Søndergaard I 《Applied microbiology and biotechnology》2011,90(6):1923-1932
Hydrophobins are small fungal proteins with amphipatic properties and the ability to self-assemble on a hydrophobic/hydrophilic
interface; thus, many technical applications for hydrophobins have been suggested. The pathogenic fungus Aspergillus fumigatus expresses the hydrophobins RodA and RodB on the surface of its conidia. RodA is known to be of importance to the pathogenesis
of the fungus, while the biological role of RodB is currently unknown. Here, we report the successful expression of both hydrophobins
in Pichia pastoris and present fed-batch fermentation yields of 200–300 mg/l fermentation broth. Protein bands of expected sizes were detected
by SDS-PAGE and western blotting, and the identity was further confirmed by tandem mass spectrometry. Both proteins were purified
using his-affinity chromatography, and the high level of purity was verified by silver-stained SDS-PAGE. Recombinant RodA
as well as rRodB were able to convert a glass surface from hydrophilic to hydrophobic similar to native RodA, but only rRodB
was able to decrease the hydrophobicity of a Teflon-like surface to the same extent as native RodA, while rRodA showed this
ability to a lesser extent. Recombinant RodA and native RodA showed a similar ability to emulsify air in water, while recombinant
RodB could also emulsify oil in water better than the control protein bovine serum albumin (BSA). This is to our knowledge
the first successful expression of hydrophobins from A. fumigatus in a eukaryote host, which makes it possible to further characterize both hydrophobins. Furthermore, the expression strategy
and fed-batch production using P. pastoris may be transferred to hydrophobins from other species. 相似文献
7.
Nearly 30% of currently approved recombinant therapeutic proteins are produced in Escherichia coli. Due to its well-characterized genetics, rapid growth and high-yield production, E. coli has been a preferred choice and a workhorse for expression of non-glycosylated proteins in the biotech industry. There is
a wealth of knowledge and comprehensive tools for E. coli systems, such as expression vectors, production strains, protein folding and fermentation technologies, that are well tailored
for industrial applications. Advancement of the systems continues to meet the current industry needs, which are best illustrated
by the recent drug approval of E. coli produced antibody fragments and Fc-fusion proteins by the FDA. Even more, recent progress in expression of complex proteins
such as full-length aglycosylated antibodies, novel strain engineering, bacterial N-glycosylation and cell-free systems further
suggests that complex proteins and humanized glycoproteins may be produced in E. coli in large quantities. This review summarizes the current technology used for commercial production of recombinant therapeutics
in E. coli and recent advances that can potentially expand the use of this system toward more sophisticated protein therapeutics. 相似文献
8.
A gene encoding the rice (Oryza sativa L.) 90-kDa heat shock protein (OsHsp90) was introduced into Escherichia coli using the pGEX-6p-3 expression vector with a glutathione-S-transferase (GST) tag to analyze the possible function of this protein under heat stress for the first time. We compared
the survivability of E. coli (BL21) cells transformed with a recombinant plasmid containing GST-OsHsp90 fusion protein with control E. coli cells transformed with the plasmid containing GST and the wild type BL21 under heat shock after isopropyl β-d-thiogalactopyranoside induction. Cells expressing GST-OsHsp90 demonstrated thermotolerance at 42, 50, and 70°C, treatments
that were more harmful to cells expressing GST and the wild type. Further studies were carried out to analyze the heat-induced
characteristics of OsHsp90 at 42, 50, and 70°C in vitro. When cell lysates from E. coli transformants were heated at these heat stresses, expressed GST-OsHsp90 prevented the denaturation of bacterial proteins
treated with 42°C heat shocks, and partially prevented that of proteins treated at 50 and 70°C; meanwhile, cells expressing
GST-OsHsp90 withstood the duration at 50°C. These results indicate that OsHsp90 functioned as a chaperone, binding to a subset
of substrates, and maintained E. coli growth well at high temperatures. 相似文献
9.
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
10.
Withu Choosri Regina Paukner Petra Wührer Dietmar Haltrich Christian Leitner 《World journal of microbiology & biotechnology》2011,27(6):1349-1353
The gene gaoA encoding the copper-dependent enzyme galactose oxidase (GAO) from Fusarium graminearum PH-1 was cloned and successfully overexpressed in E. coli. Culture conditions for cultivations in shaken flasks were optimized, and optimal conditions were found to be double-strength
LB medium, 0.5% lactose as inducer, and induction at the reduced temperature of 25°C. When using these cultivation conditions ~24 mg
of active GAO could be produced in shaken flasks per litre medium. Addition of copper to the fermentation medium decreased
the enzyme production significantly. The His-tagged recombinant enzyme could be purified conveniently with a single affinity
chromatography step. The purified enzyme showed a single band on SDS–PAGE with an apparent molecular mass of 66 kDa and had
kinetic properties similar to those of the fungal wild-type enzyme. 相似文献
11.
Two cDNA fragments (lrF1 and lrF2) representing a fibrinolytic enzyme gene of F-III-2 (GenBank AB045719), without and with
signal peptide coding sequence, were cloned from earthworm Lumbricus rubellus. The two fragments were inserted into bacterial expression vector pET28a (+), respectively. Subsequent expression showed
that both lrF1 and lrF2 proteins were produced as an inclusion body form in E. coli BL21 (DE3) pLysE. After protein refolding and purification, the fusion lrF1 and its derivative without poly histidine tags
at the N-terminus showed fibrinolytic activity on fibrin plates with relative activity of 134.3 U/mg protein and 139.7 U/mg
protein, respectively, whereas the fusion lrF2 and its derivative without the tags at the N-terminus, had no fibrinolytic
activity. The results indicated that the E. coli expression system could not recognize the endogenous signal peptide of F-III-2, and the effect of the histidine tags at the
N-terminus on the fibrinolytic activity of the expressed protein was insignificant. 相似文献
12.
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
13.
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
14.
Preeyanan Anwised Nisachon Jangpromma Theeranan Temsiripong Rina Patramanon Sakda Daduang Sarawut Jitrapakdee Tomohiro Araki Sompong Klaynongsruang 《The protein journal》2016,35(4):256-268
Recombinant Crocodylus siamensis hemoglobin (cHb) has been constructed and expressed using Escherichia coli as the expression system in conjunction with a trigger factor from the Cold-shock system as the fusion protein. While successful processing as soluble protein in E. coli was achieved, the net yields of active protein from downstream purification processes remained still unsatisfactory. In this study, cHb was constructed and expressed in the eukaryotic expression system Pichia pastoris. The results showed that cHb was excreted from P. pastoris as a soluble protein after 72 h at 25 °C. The amino acid sequence of recombinant cHb was confirmed using LC–MS/MS. Indeed, the characteristic of Hb was investigated by external heme incorporation. The UV–Vis profile showed a specific pattern of the absorption at 415 nm, indicating the recombinant cHb was formed complex with heme, resulting in active oxyhemoglobin (OxyHb). This result suggests that the heme molecules were fully combined with heme binding site of the recombinant cHb, thus producing characteristic red color for the OxyHb at 540 and 580 nm. The results revealed that the recombinant cHb was prosperously produced in P. pastoris and exhibited a property as protein–ligand binding. Thus, our work described herein offers a great potential to be applied for further studies of heme-containing protein expression. It represents further pleasing option for protein production and purification on a large scale, which is important for determination and characterization of the authenticity features of cHb proteins. 相似文献
15.
Ozaki S Imai H Iwakiri T Sato T Shimoda K Nakayama T Hamada H 《Biotechnology letters》2012,34(3):475-481
A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-β-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4′-O-β-glucoside: 3-O-β-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for
the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to
the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically
important residue. 相似文献
16.
Xiaodan Li Bing Xia Yumei Jiang Qingsong Wu Chunyan Wang Lisi He Feng Peng Ren Wang 《Molecular biology reports》2010,37(2):995-1001
A new Lycoris radiata pathogenesis-related (PR)-4 gene, LrPR4 was isolated. LrPR4 encodes a 142 amino acid protein with a predicted molecular mass of 15.43 kDa and pI of 7.56. The putative LrPR4 shows high
similarity to PR4 type proteins from various plant species and belongs to the Barwin family. Like other PR4s from monocot
plants, LrPR4 protein contains a conserved Barwin domain and has a signal peptide at its N-terminus. The recombinant LrPR4
protein expressed in Escherichia coli showed activity towards hydrolysing RNA from L. radiata bulbs and antifungal activity. The results of this study suggest that LrPR4 may play a role in the disease resistance responses
of plant against pathogen attacks though its antifungal activity. 相似文献
17.
Production of bioactive human hemangiopoietin in <Emphasis Type="Italic">Escherichia coli</Emphasis>
To devise an efficient approach for production of human hemangiopoietin (hHAPO), the gene of hHAPO was synthesized and subcloned
into the pSUMO vector with a SUMO tag at the N-terminus. The expression construct was then transformed into the expression
strain E. coli BL21(DE3). The fusion protein was expressed in soluble form and identified by SDS-PAGE and Western blotting. The fusion protein
was purified to 90% purity by metal chelate chromatography with a yield of 45 mg per liter fermentation culture. The SUMO
tag was removed by cleavage with SUMO protease at room temperature for 1 h, and the hHAPO was then re-purified by the metal
chelate chromatography. Finally, about 21 mg hHAPO was obtained from 1 liter of fermentation culture with no less than 95%
purity. The recombinant hHAPO significantly stimulated the proliferation of human umbilical vein endothelial cells. 相似文献
18.
da Silva Pinto L Gonçales RA Conceição FR Knabah PF Borsuk S Campos VF Arruda FV Leite FP 《Applied microbiology and biotechnology》2012,95(5):1235-1241
Bacillus sphaericus produces a two-chain binary toxin composed of BinA (42 kDa) and BinB (51 kDa), which are deposited as parasporal crystals during sporulation. The toxin is highly active against Culex larvae and Aedes and Anopheles mosquitoes, which are the principal vectors for the transmission of malaria, yellow fever, encephalitis, and dengue. The use of B. sphaericus and Bacillus thuringiensis in mosquito control programs is limited by their sedimentation in still water. In this study, the binA and binB genes were cloned and the recombinant BinAB protein was expressed in three strains of Escherichia coli. These recombinant strains were used in a toxicity assay against Culex quinquefasciatus larvae. The highest expression level was achieved when both proteins were expressed in a single operon construct. The BinAB protein expressed in the E. coli Arctic strain showed higher larvicidal activity than either of the recombinant proteins from the E. coli Ril or pLysS strains. Furthermore, it had the highest oviposition attraction (49.1%, P?0.05). These data suggest that biologically active recombinant BinA and BinB toxins might be useful in mosquito control programs, delivered by inactivated bacterial cells or in traps. 相似文献
19.
Proteolytic degradation is the primary obstacle in the use of the yeast Pichia pastoris for the expression of recombinant proteins. During the production of a recombinant Plasmodium falciparum circumsporozoite protein in this system, the (NANP)
n
repeats region at the N-terminus were completely proteolytically degraded. To remove the potential proteolytic site within
the recombinant protein, different strategies were tried, including adjusting the cultivation conditions and mutating the
sequence at the junction of the repeat domain and C-terminal region, but the degradation continued. However, modification
of the N-terminal sequence by adding an epitope-based peptide to the N-terminus not only protected the repeat domain from
cleavage by native proteases during longer induction in the yeast host and purification process, but also stabilized this
recombinant protein emulsified with adjuvant ISA720 for at least 6 months. The results showed that proteolytic degradation
of the recombinant circumsporozoite protein produced in P. pastoris was amino acid sequence (NANP)-specific, and that this effect was likely dependent on the conformation of the recombinant
protein. 相似文献
20.
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