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1.
New secretion vectors containing the Bacillus sp. endoxylanase signal sequence were constructed for the secretory production of recombinant proteins in Escherichia coli. The E. coli alkaline phosphatase structural gene fused to the endoxylanase signal sequence was expressed from the trc promoter in various E. coli strains by induction with IPTG. Among those tested, E. coli HB101 showed the highest efficiency of secretion (up to 25.3% of total proteins). When cells were induced with 1 mM IPTG,
most of the secreted alkaline phosphatase formed inclusion bodies in the periplasm. However, alkaline phosphatase could be
produced as a soluble form without reduction of expression level by inducing with less (0.01 mM) IPTG, and greater than 90%
of alkaline phosphatase could be recovered from the periplasm by the simple osmotic shock method. Fed-batch cultures were
carried out to examine the possibility of secretory protein production at high cell density. Up to 5.2 g/l soluble alkaline
phosphatase could be produced in the periplasm by the pH-stat fed-batch cultivation of E. coli HB101 harboring pTrcS1PhoA. These results demonstrate the possibility of efficient secretory production of recombinant proteins
in E. coli by high cell density cultivation.
Received: 8 September 1999 / Received revision: 3 January 2000 / Accepted 4 January 2000 相似文献
2.
F. Mahboudi F. Barkhordari R.M. Godarzi S. Enayati F. Davami 《Journal of applied microbiology》2013,114(2):364-372
Aims
A novel chimeric‐truncated form of tissue‐type plasminogen activator (t‐PA) with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in Escherichia coli (BL21) strain and compare the protein potency in batch and fed‐batch processes.Methods and Results
The expression cassette for the novel t‐PA was prepared in pET‐28a(+). The E. coli expression procedure was compared in traditional batch and newly developed fed batch, EnBase® Flo system. The protein was purified in soluble format, and potency results were identified using Chromolize t‐PA Assay Kit. The fed‐batch fermentation mode, coupled with a Ni‐NTA affinity purification procedure under native condition, resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46·66 IU mg?1) compared to traditional batch mode (35·8 IU mg?1).Conclusions
Considering the undeniable advantages of expression in the prokaryotic expression systems such as E. coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed‐batch cultivation methods showed the potential to replace miss‐folded formats of protein with proper folded, soluble form with improved potency.Significance and Impact of the Study
Escherichia coli expression of recombinant proteins still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post‐translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system. 相似文献3.
Wang Yueling Jiang Yan Yang De Li Wanyi Gong Tianxiang Feng Yan Jiang Zhonghua Li Mingyuan 《World journal of microbiology & biotechnology》2009,25(5):917-920
Mouse beta defensin-1 (mBD-1) is a cationic peptide with broad antimicrobial activity. The mBD-1 gene was cloned and fused with TrxA to construct pET32-mBD1, which was transformed into E. coli BL21 (DE3). The optimal expression conditions of fusion protein TrxA–mBD1 were: cultivation at 32°C in 2 × YT medium, induction
with 0.2 mM isopropylthio-d-galactoside (IPTG), and post-induction expression for 8 h. The fusion protein was highly soluble (90.0%) and accounted for
65% of the total soluble protein; and its volumetric productivity reached 0.67 g/l, i.e., 0.14 g/l of recombinant mBD-1. At
5 μM, purified recombinant mBD-1 killed 50% of Candida albicans.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
4.
Co-production of biomass and metabolites by cell retention culture of <Emphasis Type="Italic">Leuconostoc citreum</Emphasis> 总被引:1,自引:0,他引:1
Cell retention culture of lactic acid bacterium Leuconostoc citreum was carried out in a fermentor equipped with an internal ceramic filtration system to co-produce biomass and metabolites.
The filtration system was composed of porous ceramic filter module with pore size of 0.1 μm and total surface area of 330 cm2. High cell density cultivation of L. citreum was achieved within the fermentor, while extracellular metabolites such as mannitol and d-lactic acid were produced through the filter with high productivities. In batch culture of L. citreum using a medium containing 50 g/L of glucose and 100 g/L of fructose, the maximum optical density (OD) monitored at 660 nm
was 13 with 65 g/L of mannitol and 38 g/L of lactic acid. In cell retention culture of L. citreum with dilution rate of 0.07 h−1, OD increased to 75, which was 6 times higher than that in batch culture. The concentrations of mannitol and lactic acid
increased to 85 and 45 g/L, respectively, and were maintained throughout the cultivation to 105 h. By increasing dilution
rate to 0.13 h−1, the productivities of mannitol and lactic acid increased to 8.5 and 4.2 g/L/h, respectively, which were 2.7 to 3 times higher
than those in batch culture, suggesting that cell retention culture using internal filtration system is highly effective for
co-production of useful cell biomass and various extracellular metabolites. 相似文献
5.
Thi Thuy Tran Gashaw Mamo Bo Mattiasson Rajni Hatti-Kaul 《Journal of industrial microbiology & biotechnology》2010,37(3):279-287
Phytase is used as a feed additive for degradation of antinutritional phytate, and the enzyme is desired to be highly thermostable
for it to withstand feed formulation conditions. A Bacillus sp. MD2 showing phytase activity was isolated, and the phytase encoding gene was cloned and expressed in Escherichia coli. The recombinant phytase exhibited high stability at temperatures up to 100°C. A higher enzyme activity was obtained when
the gene expression was done in the presence of calcium chloride. Production of the enzyme by batch- and fed-batch cultivation
in a bioreactor was studied. In batch cultivation, maintaining dissolved oxygen at 20–30% saturation and depleting inorganic
phosphate below 1 mM prior to induction by IPTG resulted in over 10 U/ml phytase activity. For fed–batch cultivation, glucose
concentration was maintained at 2–3 g/l, and the phytase expression was increased to 327 U/ml. Induction using lactose during
fed-batch cultivation showed a lag phase of 4 h prior to an increase in the phytase activity to 71 U/ml during the same period
as IPTG-induced production. Up to 90% of the total amount of expressed phytase leaked out from the E. coli cells in both IPTG- and lactose-induced fed-batch cultivations. 相似文献
6.
Low KO Mahadi NM Rahim RA Rabu A Abu Bakar FD Murad AM Illias RM 《Journal of industrial microbiology & biotechnology》2011,38(9):1587-1597
Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific
activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette)
transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design
and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular
recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving
six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant
CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted
extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml,
resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of
about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient
protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage. 相似文献
7.
The gene encoding the cyclodextrin glucanotransferase of Paenibacillus pabuli US132 was connected to the amylase signal peptide of Bacillus stearothermophilus. This leads to an efficient secretion of the recombinant enzyme into the culture medium of Escherichia coli as an active form contrasting with the native construction leading to a periplasmic production. The optimum cultivation conditions
for the maximum expression were optimized, using a Box-Behnken design under the response surface methodology, and found to
be a post-induction temperature of 24°C, an induction-starting A600 nm of 0.85, an isopropyl-β-D-thiogalactopyranoside level of 0.045 mM and a post-induction time of 3.9 h. The screening of media components and their
concentration were achieved using a Plackett-Burman and a Box-Behnken designs sequentially. Under the optimized conditions
selected and in agreement with the predicted model, an activity of 6.03 U/mL was attained. This CGTase production was three-times
higher than that using the non-optimized culture conditions (2 U/mL). 相似文献
8.
In batch cultivation, growth of a recombinant Escherichia coli with an inducible T7 expression system and maximum expression of a bioadhesive precursor (BP) protein was similar in the strains with and without the plasmid vector, pLysS. In fed-batch cultivation, however, the strain harboring pLysS grew slower and had a lower level of BP protein expression than that obtained with the strain without pLysS. This suggests that the presence of pLysS in the T7 expression system strongly affects the cell growth and expression of BP protein in high cell density cultivation. 相似文献
9.
Carla Oliveira Sofia Costa José A. Teixeira Lucília Domingues 《Molecular biotechnology》2009,43(3):212-220
cDNA clones encoding frutalin, the α-d-galactose-binding lectin expressed in breadfruit seeds (Artocarpus incisa), were isolated and sequenced. The deduced amino acid sequences indicated that frutalin may be encoded by a family of genes.
The NCBI database searches revealed that the frutalin sequence is highly homologous with jacalin and mornigaG sequences. Frutalin
cDNA was re-amplified and cloned into the commercial expression vector pET-25b(+) for frutalin production in Escherichia coli. An experimental factorial design was employed to maximise the soluble expression of the recombinant lectin. The results
indicated that temperature, time of induction, concentration of IPTG and the interaction between the concentration of IPTG
and the time of induction had the most significant effects on the soluble expression level of recombinant frutalin. The optimal
culture conditions were as follows: induction with 1 mM IPTG at 22°C for 20 h, yielding 16 mg/l of soluble recombinant frutalin.
SDS-PAGE and Western blot analysis revealed that recombinant frutalin was successfully expressed by bacteria with the expected
molecular weight (17 kDa). These analyses also showed that recombinant frutalin was mainly produced as insoluble protein.
Recombinant frutalin produced by bacteria revealed agglutination properties and carbohydrate-binding specificity similar to
the native breadfruit lectin. 相似文献
10.
Lüders S David F Steinwand M Jordan E Hust M Dübel S Franco-Lara E 《Applied microbiology and biotechnology》2011,91(1):81-90
Bacillus megaterium was used for production of the lysozyme-specific recombinant scFv D1.3 antibody fragment. Key process parameters like the
temperature and the hydromechanical stress play a very important role for significant product formation during process development
or scale-up. In this study, the influence of these two variables on growth and recombinant antibody fragment production in
a 2-L lab-scale bioreactor system was investigated using a central composite design. Especially a significant influence of
the hydromechanical stress on antibody fragment production was detected in batch cultivations. While volumetric power inputs
of about 0.5 kW/m3 (agitation rates around 500 min−1) are usually employed in batch cultivations, in this work maximal product concentration was found at a volumetric power input
of about 0.06 kW/m3 (agitation rate around 250 min−1) and at a high cultivation temperature of 41 °C. The influence of the two process variables at single-cell level was estimated
using flow cytometry too. The characterization was done by estimating the membrane potential giving a hint on bioprocess productivity
and secretion capability: the best production was obtained through big cells with low specific membrane potential, which grew
at low volumetric power inputs and high cultivation temperatures. 相似文献
11.
Parallel operated milliliter-scale stirred tank bioreactors were applied for recombinant protein expression studies in simple
batch experiments without pH titration. An enzymatic glucose release system (EnBase), a complex medium, and the frequently
used LB and TB media were compared with regard to growth of Escherichia coli and recombinant protein expression (alcohol dehydrogenase (ADH) from Lactobacillus brevis and formate dehydrogenase (FDH) from Candida boidinii). Dissolved oxygen and pH were recorded online, optical densities were measured at-line, and the activities of ADH and FDH
were analyzed offline. Best growth was observed in a complex medium with maximum dry cell weight concentrations of 14 g L−1. EnBase cultivations enabled final dry cell weight concentrations between 6 and 8 g L−1. The pH remained nearly constant in EnBase cultivations due to the continuous glucose release, showing the usefulness of
this glucose release system especially for pH-sensitive bioprocesses. Cell-specific enzyme activities varied considerably
depending on the different media used. Maximum specific ADH activities were measured with the complex medium, 6 h after induction
with IPTG, whereas the highest specific FDH activities were achieved with the EnBase medium at low glucose release profiles
24 h after induction. Hence, depending on the recombinant protein, different medium compositions, times for induction, and
times for cell harvest have to be evaluated to achieve efficient expression of recombinant proteins in E. coli. A rapid experimental evaluation can easily be performed with parallel batch operated small-scale stirred tank bioreactors. 相似文献
12.
原动蛋白2(PK2)是近年发现的一个具有多种生物学功能的蛋白质因子。采用Design-Expert 7.0.0软件对重组PK2表达的最优诱导条件进行响应面分析。结果表明,诱导起始时机与诱导物浓度是影响重组PK2表达水平的显著性因子。同时,诱导时间-诱导时机、诱导时机-诱导物浓度之间的相互作用对PK2的表达水平具有显著性影响。试验数据拟合与极值分析表明最优诱导条件为:工程菌OD600为0.60时,采用终浓度为0.62 mmol/L的IPTG在37℃诱导培养2.82 h,此时重组PK2的表达水平为170.73 mg/L。优化后的诱导条件导致重组PK2的表达水平提高了51.1%,而所需IPTG减少38.0%,诱导时间缩短53.0%,有利于大量制备重组PK2进行后续功能研究及应用研究。 相似文献
13.
Microalgal lipids may be a more sustainable biodiesel feedstock than crop oils. We have investigated the potential for using
the crude glycerol as a carbon substrate. In batch mode, the biomass and lipid concentration of Chlorella protothecoides cultivated in a crude glycerol medium were, respectively, 23.5 and 14.6 g/l in a 6-day cultivation. In the fed-batch mode,
the biomass and lipid concentration improved to 45.2 and 24.6 g/l after 8.2 days of cultivation, respectively. The maximum
lipid productivity of 3 g/l day in the fed-batch mode was higher than that produced by batch cultivation. This work demonstrates
the feasibility of crude biodiesel glycerol as an alternative carbon substrate to glucose for microalgal cultivation and a
cost reduction of carbon substrate feed in microalgal lipid production may be expected. 相似文献
14.
Khalilzadeh R Mohammadian-Mosaabadi J Bahrami A Nazak-Tabbar A Nasiri-Khalili MA Amouheidari A 《Journal of industrial microbiology & biotechnology》2008,35(12):1643-1650
The fed-batch process using glucose as the sole source of carbon and energy with exponential feeding rate was carried out
for high cell density cultivation of recombinant Escherichia coli BL21 (DE3) expressing human granulocyte-colony stimulating factor (hG-CSF). IPTG was used to induce the expression of hG-CSF
at 48 g dry cell wt l−1 during high cell density culture of recombinant E. coli BL21 (DE3) [pET23a-g-csf]. The final cell density, specific yield and overall productivity of hG-CSF were obtained as ~64 g
dry cell wt l−1, 223 mg hG-CSF g−1 dry cell wt and 775 mg hG-CSF l−1 h−1, respectively. The resulting purification process used cell lysis, inclusion body (IB) preparation, refolding, DEAE and Butyl-Sepharose.
Effects of different process conditions such as cell lysis and washing of IB were evaluated. The results reveal that the cells
lyzed at 1,200 bar, 99.9% and Triton removed about 64% of the LPS but sarcosyl had no effect on removal of nucleic acids and
LPS. Further analysis show that DEAE column removes DNA about 84%. Cupper concentration was identified as parameter that could
have a significant impact on aggregation, as an unacceptable pharmaceutical form that decrease process yields. The purity
of purified hG-CSF was more than 99%. Also the comparison of activity between purified hG-CSF and commercial form do not show
valuable decrease in activity in purified form. 相似文献
15.
Peter M. Kilonzo Argyrios Margaritis Maurice A. Bergougnou 《Journal of industrial microbiology & biotechnology》2010,37(8):773-783
The recombinant Saccharomyces cerevisiae strain C468/pGAC9 has an unstable hybrid plasmid pGAC9, which directs production of glucoamylase. A fibrous cotton material
with a good adsorption capability for recombinant S. cerevisiae cells was used as the immobilization matrix in an internal loop airlift-driven fibrous bed bioreactor (ILALFBB) system. With
batch cultures in the ILALFBB, the fraction of plasmid-carrying cells was 72% after more than 2 days cultivation, which was
two times higher than that in the conventional free-cell culture. Correspondingly, a high activity of glucoamylase (GA; 113 U/l)
was achieved with a high productivity of 43 U/l/h. The ILALFBB system also maintained a high fraction of viable plasmid-carrying
of 74% for glucoamylase production during repeated-batch cultures, achieving a high glucoamylase activity of 140 U/l with
a productivity of 19–130 U/l/h in all 14 batches studied during 19.8 days. The stable and long-term glucoamylase production
from the ILALFBB was attributed to the effect of cell immobilization on plasmid stability. Plasmid-carrying cells were preferentially
retained in the fibrous matrix because of their ability to adhere to the fiber surface and to form cell aggregates higher
than those of plasmid-free cells. The repeated batch using immobilized cell of recombinant S. cerevisiae in the ALALFBB system thus provides a feasible method for stable, long-term and high-level production of glucoamylase. 相似文献
16.
Liangfan Zhou Zhihui Zhao Baocun Li Yufeng Cai Shuangquan Zhang 《Protein expression and purification》2009,64(2):225-230
Antimicrobial peptide CM4, a small cationic linear α-helical peptide that consists of 35 amino acids, was isolated from Bombyx mori. To improve the expression level of CM4 in Escherichia coli, tandem repeats of CM4 gene were constructed and expressed as fusion proteins (TrxA-nCM4, n = 1, 2, 3,…,8) by constructing the vectors of pET32-nCM4 (n = 1, 2, 3,…,8). Comparison among the expression levels of soluble fusion protein TrxA-nCM4 (n = 1, 2, 3,…,8) suggested that BL21 (DE3)/pET32-3CM4 was an ideal recombinant strain for CM4 production. Under the selected conditions of cultivation and isopropylthiogalactoside (IPTG) induction, the expression level of CM4 was as high as 68 mg/l with about 21% of fusion protein in soluble form, which was the highest yield of CM4 reported so far. 相似文献
17.
Harper CB Martin S Nguyen TH Daniels SJ Lavidis NA Popoff MR Hadzic G Mariana A Chau N McCluskey A Robinson PJ Meunier FA 《The Journal of biological chemistry》2011,286(41):35966-35976
The botulinum neurotoxins (BoNTs) are di-chain bacterial proteins responsible for the paralytic disease botulism. Following binding to the plasma membrane of cholinergic motor nerve terminals, BoNTs are internalized into an endocytic compartment. Although several endocytic pathways have been characterized in neurons, the molecular mechanism underpinning the uptake of BoNTs at the presynaptic nerve terminal is still unclear. Here, a recombinant BoNT/A heavy chain binding domain (Hc) was used to unravel the internalization pathway by fluorescence and electron microscopy. BoNT/A-Hc initially enters cultured hippocampal neurons in an activity-dependent manner into synaptic vesicles and clathrin-coated vesicles before also entering endosomal structures and multivesicular bodies. We found that inhibiting dynamin with the novel potent Dynasore analog, Dyngo-4a(TM), was sufficient to abolish BoNT/A-Hc internalization and BoNT/A-induced SNAP25 cleavage in hippocampal neurons. Dyngo-4a also interfered with BoNT/A-Hc internalization into motor nerve terminals. Furthermore, Dyngo-4a afforded protection against BoNT/A-induced paralysis at the rat hemidiaphragm. A significant delay of >30% in the onset of botulism was observed in mice injected with Dyngo-4a. Dynamin inhibition therefore provides a therapeutic avenue for the treatment of botulism and other diseases caused by pathogens sharing dynamin-dependent uptake mechanisms. 相似文献
18.
A set of mutations in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) was used to create Escherichia coli strains with a reduced uptake rate of glucose. This allows a growth restriction, which is controlled on cellular rather than
reactor level, which is typical of the fed-batch cultivation concept. Batch growth of the engineered strains resulted in cell
accumulation profiles corresponding to a growth rate of 0.78, 0.38 and 0.25 h−1, respectively. The performance of the mutants in batch cultivation was compared to fed-batch cultivation of the wild type
cell using restricted glucose feed to arrive at the corresponding growth profiles. Results show that the acetate production,
oxygen consumption and product formation were similar, when a recombinant product was induced from the lacUV5 promoter. Ten times more cells could be produced in batch cultivation using the mutants without the growth detrimental production
of acetic acid. This allows high cell density production without the establishment of elaborate fed-batch control equipment.
The technique is suggested as a versatile tool in high throughput multiparallel protein production but also for increasing
the number of experiments performed during process development while keeping conditions similar to the large-scale fed-batch
performance. 相似文献
19.
《Biochemical Engineering Journal》2007,33(1):34-41
The effects of post-induction nutrient feed rates, on recombinant streptokinase production in fed-batch processes, were investigated using various feed profiles. Recombinant streptokinase was produced using a secretory expression system and was induced by a temperature up-shift, using a temperature-sensitive λPL promoter. The specific growth rates decreased sharply upon induction of recombinant protein expression, thus necessitating a variable feed strategy in the post-induction phase. The various feed profiles employed in the post-induction phase included constant feed rates, linearly increasing feed rate and exponentially varying feed rates. Significant differences were obtained in the specific activity of streptokinase produced in these fed-batch processes. A maximum activity per unit biomass of 4.96 × 106 and 4.43 × 106 IU/g DCW was achieved for exponentially decreasing feed and linearly increasing feed, respectively. The decrease in specific growth rate during the post-induction phase was also less pronounced in these cases in comparison to other fed-batch experiments. It was observed that streptokinase productivity is governed by the nutrient feed rate per unit biomass at a critical juncture after induction. The highest activity per unit biomass was obtained when the nutrient feed rate per unit biomass was around 0.7–0.8 g glucose (g DCW)−1 h−1, between 2 and 4 h after induction. 相似文献
20.
The evolution of CO2 in a fed-batch culture of recombinant Escherichia coli containing human-like collagen (HLC) cDNA was determined with an O2-enriched air supply (40%, v/v) in a 12.8 l fermentor; a maximum CO2 concentration of 12.7% in the effluent gas was detected. The CO2 pulse injection experiments showed that: (1) a 20% CO2 pulse introduced in the batch cultivation phases inhibited cell growth but if introduced in the fed-batch cultivation phases
slightly stimulated growth; and (2) CO2 inhibited HLC expression only in the expression phase, where the final HLC concentration decreased by 34% under a 3 h 20%
CO2 pulse. The higher the CO2 concentration and/or the longer the duration of the CO2 pulse, the stronger the stimulatory or inhibitory effects.
An erratum to this article can be found at 相似文献