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1.
2.
IPTG can replace lactose in auto‐induction media to enhance protein expression in batch‐cultured Escherichia coli
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Auto‐induction media containing glucose, lactose, and glycerol are a simple and efficient approach for high‐throughput protein expression in Escherichia coli with lac‐derived expression systems. Its principle is based on inducer exclusion between glucose and lactose, preventing the induction by lactose before the depletion of glucose. Isopropyl‐β‐d ‐1‐thiogalactopyranoside (IPTG)—at least in typically used millimolar concentrations—is thought to be unsuitable for this purpose since it can enter the cell by diffusion independently of inducer exclusion. In this study, using parallel batch cultivations in stirred‐tank bioreactors on a milliliter scale, we show that the induction by micromolar concentrations of IPTG is prevented in the presence of glucose. With up to 40 μM IPTG, full induction and heterologous protein expression start only after the depletion of glucose. Thus, auto‐induction is possible with either lactose or IPTG, and the expression greatly depends on the type and concentration of the inducer. The best expression of enhanced green fluorescent protein was achieved with 40 μM IPTG in stirred‐tank bioreactors on a milliliter scale. The IPTG‐based auto‐induction was also reproduced in shaking flasks. Therefore, IPTG can be used in auto‐induction media for protein expression in batch‐cultured E. coli. Furthermore, we show that acetate or arabinose can have significant effects on the auto‐induction mechanism. 相似文献
3.
This study developed an artificial neural network (ANN) to estimate the growth of microorganisms during a fermentation process.
The ANN relies solely on the cumulative consumption of alkali and the buffer capacity, which were measured on-line from the
on/off control signal and pH values through automatic pH control. The two input variables were monitored on-line from a series
of different batch cultivations and used to train the ANN to estimate biomass. The ANN was refined by optimizing the network
structure and by adopting various algorithms for its training. The software estimator successfully generated growth profiles
that showed good agreement with the measured biomass of separate batch cultures carried out between at 25 and 35_C. 相似文献
4.
Online measurement of the respiratory activity in shake flasks enables the identification of cultivation phases and patterns indicating recombinant protein production in various Escherichia coli host strains
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Nina Ihling Natalie Bittner Sylvia Diederichs Maximilian Schelden Anna Korona Georg Theo Höfler Alexander Fulton Karl‐Erich Jaeger Kohsuke Honda Hisao Ohtake Jochen Büchs 《Biotechnology progress》2018,34(2):315-327
Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7‐RNA polymerase‐dependent Escherichia coli expression strains was compared under inducing and noninducing conditions. As model enzymes, a lipase A from Bacillus subtilis (BSLA) and a 3‐hydroxybutyryl‐CoA dehydrogenase from Thermus thermophilus (HBD) were chosen. Four strains were compared during expression of both enzymes in autoinduction medium. Additionally, four strains were compared during expression of the BSLA with IPTG induction. It was found that the metabolic burden during recombinant protein production induces a phase of constant OTR, while undisturbed cell growth with no or little product formation is indicated by an exponential increase. This pattern is independent of the host strain, expressed enzyme, and induction method. Furthermore, the OTR gives information about carbon source consumption, biomass formation, and the transition from production to noninduced second growth phase, thereby ensuring a fair comparison of different strains. In conclusion, online monitoring of the respiration activity is suited to qualitatively identify, if a recombinant protein is produced by a strain or not. Furthermore, laborious offline sampling is avoided. Thus, the technique is easier and faster compared to conventional approaches. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:315–327, 2018 相似文献
5.
《Biochemical Engineering Journal》2006,30(3):235-242
Fed-batch production of recombinant fuculose-1-phosphate aldolase (FucA) by Escherichia coli XL1 Blue MRF′ (pTrcfuc) has been automated by using a simple feedback specific growth rate control strategy. Non-induced continuous cultures were conducted in order to characterize substrate consumption and carbon dioxide production yields and rates. In fed-batch cultures, substrate feeding rate was adjusted using on-line biomass estimation based on exhaust gas analysis and macroscopic mass balances. Overexpression of recombinant protein induced by isopropyl-β-d-thiogalactopyranoside (IPTG) under trc promoter did not affect significantly the control of specific growth rate during 7 h after induction. Growth and protein production curves were parallel until high level of protein expression started to inhibit cell growth. The proposed specific growth rate control strategy has been successfully applied to both non-induced and induced fed-batch cultures that do not exhibit severe growth rate depression. 相似文献
6.
Bong Hyun Chung Dong Jin Seo Young Hoon Park Sun Bok Lee Moon Hi Han 《Biotechnology Techniques》1991,5(3):163-168
Summary The amount of alkali added to the fermentation broth of a recombinant Escherichia coli strain for pH control was monitored on-line by an electronic balance interfaced to a computer. It was successfully correlated with the cell mass, and consequently the cell growth could be well estimated. Using this cell growth estimation technique, an automatic temperature induction was successfully carried out to produce human interleukin-2 in a high yield from E. coli harbouring a temperature controlled expression vector system. 相似文献
7.
研究用乳糖替代IPTG作为诱导剂进行重组蛋白的表达,观察乳糖对乳糖操纵子调控的基因工程菌发酵及重组血管内皮抑素表达的影响,从而选取最佳诱导表达条件。以重组人血管内皮抑素表达工程菌pETrhEN/BL21(DE3)作为研究对象,分别用IPTG和乳糖作为诱导剂,在摇瓶中进行表达实验。并对重组蛋白质表达量进行分析。然后在5 L发酵罐中进行验证。在摇瓶培养条件下,乳糖浓度大于0.5 g/L即可以诱导目的蛋白的表达。乳糖浓度1 g/L时诱导目的蛋白表达量与1 mmol/L的IPTG相当,当乳糖浓度为10 g/L,目的蛋白表达量达到最大。在发酵罐培养条件下,补料4 h后葡萄糖浓度基本耗尽,此时开始加入乳糖。诱导后1 h,即有重组蛋白表达,在诱导后4 h达到高峰(占菌体可溶性蛋白的56%),与此同时,诱导后5 h菌体浓度也达到最高值。在以乳糖操纵子为调控手段的工程菌表达系统中,可以使用乳糖作为诱导剂,诱导应在葡萄糖消耗完后进行。 相似文献
8.
On-line characterization of a hybridoma cell culture process 总被引:2,自引:0,他引:2
The on-line determination of the physiological state of a cell culture process requires reliable on-line measurements of various parameters and calculations of specific rates from these measurements. The cell concentration of a hybridoma culture was estimated on-line by measuring optical density (OD) with a laser turbidity probe. The oxygen uptake rate (OUR) was determined by monitoring dynamically dissolved oxygen concentration profiles and closing oxygen balances in the culture. The base addition for neutralizing lactate produced by cells was also monitored on-line via a balance. Using OD and OUR measurements, the specific growth and specific oxygen consumption rates were determined on-line. By combining predetermined stoichiometric relationships among oxygen and glucose consumption and lactate production, the specific glucose consumption and lactate production rates were also calculated on-line. Using these on-line measurements and calculations, the hybridoma culture process was characterized on-line by identifying the physiological states. They will also facilitate the implementation of nutrient feeding strategies for fed-batch and perfusion cultures. (c) 1994 John Wiley & Sons, Inc. 相似文献
9.
M. Pokkinen Z. R. Flores Bustamante H. Asama I. Endo P. Linko 《Bioprocess and biosystems engineering》1992,7(7):319-323
The on-line calculated specific rates of growth, substrate consumption and product formation were used to diagnose microbial activities during a lactic acid fermentation. The specific rates were calculated from on-line measured cell mass, and substrate and product concentrations. The specific rates were more sensitive indicators of slight changes in fermentation conditions than such monitored data as cell mass or product concentrations.List of Symbols
1/h
specific rate of cell growth
-
1/h
specific rate of substrate consumption
-
1/h
specific rate of product formation
-
*
dimensionless specific rate of cell growth
-
*
dimensionless specific rate of substrate consumption
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*
dimensionless specific rate of product formation
-
max 1/h
maximum specific rate of cell growth
-
max 1/h
maximum specific rate of substrate consumption
-
max 1/h
maximum specific rate of product formation
-
X g/l
cell mass concentration
-
S g/l
substrate concentration
-
S
*
dimensionless substrate concentration
-
S
0 g/l
initial substrate concentration
-
P g/l
product concentration 相似文献
10.
Luan Tales Costa de Paiva Vasconcelos Marcos Antônio Oliveira Filho Vitor Troccoli Ribeiro Jaciara Silva de Araújo Francisco Canindé de Sousa Junior Daniella Regina Arantes Martins 《Preparative biochemistry & biotechnology》2013,43(10):968-976
AbstractLeishmaniosis is a complex of diseases that can be fatal, if not given proper attention. Despite its relevance in the public health system, there is no vaccine capable of preventing the disease in humans so far and its treatment is expensive and aggressive to human health. The present study aims to optimize the induction parameters of the 503 Leishmania i. chagasi antigen expressed in recombinant Escherichia coli M15. The induction at different cell densities was evaluated in order to analyze the influence of the induction time on the yield of the protein of interest. In this segment, lactose and isopropyl-β-d-thiogalactopyranoside (IPTG) were used as inducer molecules, using various concentrations: 0.1?g/L, 1.0?g/L, and 10?g/L for lactose and 20?μM, 100?μM, 500?μM, and 1000?μM for IPTG. The results presented that the concentration of IPTG that obtained the higher antigen levels was that of 100?μM (0.087?g/L), a 10-fold lower concentration than was being previously used in this type of system and for lactose, it was 1?g/L (0.016?g/L). Thus, the induction with 100?μM allowed obtaining the antigen with a concentration 5.6 times higher than the lactose induction maximum concentration. 相似文献
11.
Jennifer O. Shitu John M. Woodley Richard Wnek Michel Chartrain Christopher J. Hewitt 《Biotechnology letters》2009,31(4):577-584
The expression of interleukin-13 (IL13) following induction with IPTG in Escherichia coli results in metabolic changes as indicated by multi-parameter flow cytometry and traditional methods of fermentation profiling
(O2 uptake rate, CO2 evolution rate and optical density measurements). Induction early in the rapid growth phase was optimal although this led
to lower overall biomass concentrations and lower maximum specific growth rates. In contrast, induction in the mid-rapid growth
phase was the most detrimental to cell quality as measured by cytoplamsic membrane depolarisation. 相似文献
12.
Increasing Secretion of a Bivalent Anti-T-Cell Immunotoxin by Pichia pastoris 总被引:2,自引:0,他引:2
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Jung Hee Woo Yuan Yi Liu Scott Stavrou David M. Neville Jr. 《Applied microbiology》2004,70(6):3370-3376
The bivalent anti-T-cell immunotoxin A-dmDT390-bisFv(G4S) was developed for treatment of T-cell leukemia and autoimmune diseases and for tolerance induction for transplantation. This immunotoxin was produced extracellularly in toxin-sensitive Pichia pastoris JW102 (Mut+) under control of the AOX1 promoter. There were two major barriers to efficient immunotoxin production, the toxicity of the immunotoxin for P. pastoris and the limited capacity of P. pastoris to secrete the immunotoxin. The immunotoxin toxicity resulted in a decrease in the methanol consumption rate, cessation of cell growth, and low immunotoxin productivity after the first 22 h of methanol induction. Continuous cell growth and continuous immunotoxin secretion after the first 22 h of methanol induction were obtained by adding glycerol to the methanol feed by using a 4:1 methanol-glycerol mixed feed as an energy source and by continuously adding a yeast extract solution during methanol induction. The secretory capacity was increased from 22.5 to 37 mg/liter by lowering the induction temperature. A low temperature reduced the methanol consumption rate and protease activity in the supernatant but not cell growth. The effects of adding glycerol and yeast extract to the methanol feed were synergistic. Adding yeast extract primarily enhanced methanol utilization and cell growth, while adding glycerol primarily enhanced immunotoxin production. The synergy was further enhanced by decreasing the induction temperature from 23 to 15°C, which resulted in a robust process with a yield of 37 mg/liter, which was sevenfold greater than the yield previously reported for a toxin-resistant CHO cell expression system. This methodology should be applicable to other toxin-related recombinant proteins in toxin-sensitive P. pastoris. 相似文献
13.
Robert Huber Thomas G Palmen Nadine Ryk Anne-Kathrin Hillmer Karina Luft Frank Kensy Jochen Büchs 《BMC biotechnology》2010,10(1):22
Background
High-throughput cultivations in microtiter plates are the method of choice to express proteins from recombinant clone libraries. Such processes typically include several steps, whereby some of them are linked by replication steps: transformation, plating, colony picking, preculture, main culture and induction. In this study, the effects of conventional replication methods and replication tools (8-channel pipette, 96-pin replicators: steel replicator with fixed or spring-loaded pins, plastic replicator with fixed pins) on growth kinetics of Escherichia coli SCS1 pQE-30 pSE111 were observed. Growth was monitored with the BioLector, an on-line monitoring technique for microtiter plates. Furthermore, the influence of these effects on product formation of Escherichia coli pRhotHi-2-EcFbFP was investigated. Finally, a high-throughput cultivation process was simulated with Corynebacterium glutamicum pEKEx2-phoD-GFP, beginning at the colony picking step. 相似文献14.
研究以乳糖代替IPTG作为诱导剂诱导重组人胸腺肽α1表达的可行性,对乳糖诱导的时机、乳糖浓度、诱导持续时间以及其它诱导条件进行研究,确定了乳糖诱导的最佳条件。结果表明,乳糖能有效地诱导重组人胸腺肽α1的表达,并且目的蛋白的表达量略高于IPTG的诱导量。 相似文献
15.
Qingbao Ding Ling Ou Dongzhi Wei Xiaokun Wei 《Nucleosides, nucleotides & nucleic acids》2013,32(5):360-368
Recombinant E. coli pDEOA was constructed and lactose can be used instead of IPTG to induce the expression of thymidine phosphorylase by pDEOA. The use of lactose at concentrations higher than 0.5 mmol/L had an induction effect similar to that of IPTG but resulted in a longer initial induction time and better cell growth. The thymidine phosphorylase induced by lactose was very stable at 50°C. Intact pDEOA cells induced by lactose can be used as a source of thymidine phosphorylase. Under standard reaction conditions, several deoxynucleosides were effectively produced from thymidine. 相似文献
16.
Michele Galluccio Lorena Pochini Linda Amelio Rosita Accardi Massimo Tommasino Cesare Indiveri 《Protein expression and purification》2009,68(2):215-220
The hOCTN1 amplified from skin fibroblast RNA was cloned in pET-28a(+) or in pH6EX3 plasmid. The encoded recombinant hOCTN1 resulted in a 6-His tagged fusion protein with a 34 or 21 amino acid extra N-terminal sequence in the pET-28a(+)-hOCTN1 or in the pH6EX3-hOCTN1 constructs, respectively. Both constructs were used to express the hOCTN1 in Escherichia coli Rosetta(DE3)pLysS. The best over-expression was obtained with the pH6EX3-hOCTN1 after 6 h of induction with IPTG at 28 °C. The expressed protein with an apparent molecular mass of 54 kDa, was collected in the insoluble fraction of the cell lysate. Further improvement was obtained using the E. coli RosettaGami2(DE3)pLysS strain to express the protein encoded by pH6EX3-hOCTN1. After 6 h of induction with IPTG at 28 °C, hOCTN1 accounted for 30% of the total protein in the insoluble pellet. This protein fraction was washed with Triton X-100 and deoxycholate, solubilized with a buffer containing 0.8% Sarkosyl, 3 M urea and applied to a Ni2+-chelating chromatography column. The homogeneously purified hOCTN1 was eluted with a buffer containing 50 mM imidazole, 0.1% Triton X-100 and 50 mM 2-mercaptoethanol. A yield of about 3 mg purified protein per liter of cell culture was obtained. 相似文献
17.
Haishan Tian Lu Tang Yi Wang Xiaojie Wang Lili Guan Jian Zhang Xiaoping Wu Xiaokun Li 《International journal of peptide research and therapeutics》2011,17(2):123-129
Recombinant human keratinocyte growth factor-2 (rhKGF-2) has previously been expressed in Escherichia coli using isopropyl-β-d-thiogalactopyranoside (IPTG), a non-metabolizable and expensive compound, as the inducer. In order to determine whether IPTG
could be replaced with the cheap and natural lactose to induce rhKGF-2 expression, we examined the expression of rhKGF-2 in
flask culture and 30-l fermentation using lactose as the inducer. The optimized fermentation induced with lactose resulted
in 1,382 g of cell mass, corresponding to a 84% enhancement in cell mass compared with IPTG induction. While the expression
level of rhKGF-2 induced with lactose was comparable to that induced with IPTG, the solubility of target protein was increased
by lactose induction than by IPTG induction. The recombinant protein was further purified by cation exchange and heparin-affinity
chromatography. 255 milligrams of pure rhKGF-2 was achieved per liter culture by lactose induction, 52% higher than that obtained
by IPTG induction. A preliminary biochemical characterization of purified rhKGF-2 was performed by Western blotting and mitogenic
activity analysis, and the results demonstrated that the purified lactose-induced rhKGF-2 could react with anti-human KGF-2
antibody and stimulate the proliferation of FGFR2-IIIb-transfected mouse BaF3 cells as IPTG-induced rhKGF-2 could do. 相似文献
18.
The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, using on-line off-gas analyses to measure the oxygen and carbon dioxide consumption rates
continuously. A cell suspension from continuous cultures at steady state was used as the inoculum. It was observed that a
dynamic phase occurred in the initial phase of the experiment. In this phase the bacterial ferrous iron oxidation and growth
were uncoupled. After about 16 h the bacteria were adapted and achieved a pseudo-steady state, in which the specific growth
rate and oxygen consumption rate were coupled and their relationship was described by the Pirt equation. In pseudo-steady
state, the growth and oxidation kinetics were accurately described by the rate equation for competitive product inhibition.
Bacterial substrate consumption is regarded as the primary process, which is described by the equation for competitive product
inhibition. Subsequently the kinetic equation for the specific growth rate, μ, is derived by applying the Pirt equation for bacterial substrate consumption and growth. The maximum specific growth rate,
μ
max, measured in the batch culture agrees with the dilution rate at which washout occurs in continuous cultures. The maximum
oxygen consumption rate, q
O2,max, of the cell suspension in the batch culture was determined by respiration measurements in a biological oxygen monitor at
excess ferrous iron, and showed changes of up to 20% during the course of the experiment. The kinetic constants determined
in the batch culture slightly differ from those in continuous cultures, such that, at equal ferric to ferrous iron concentration
ratios, biomass-specific rates are up to 1.3 times higher in continuous cultures.
Received: 8 February 1999 / Accepted: 17 February 1999 相似文献
19.
20.
During continuous alcoholic fermentation, Saccharomyces cerevisiae 38A floc concentration was monitored using an on-line impedance probe. Since the sensor response is linear and does not depend significantly on yeast particle size, an automatic technique of determining the yeast growth rate has been developed and validated against the conventional mass balance method. 相似文献