scholarly journals Boosting Auto-induction of Recombinant Proteins in Escherichia coli with Glucose and Lactose Additives

2021 ◽  
Vol 28 ◽  
Author(s):  
Nariyasu Tahara ◽  
Itaru Tachibana ◽  
Kazuyo Takeo ◽  
Shinji Yamashita ◽  
Atsuhiro Shimada ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Culture Medium ◽  
Thermus Thermophilus ◽  
Medium Composition ◽  
Final Concentration ◽  
Induction Medium ◽  
Lac Operon ◽  
Escherichia Coli Expression ◽  
Complex Culture

Background: Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level. Objectives: In this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium. Method: First, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7lac promoter-based expression). Results: Auto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture. Conclusion: The medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.

scholarly journals Efficiency of using different types induction culture medium for hexaploid triticale anther cultivation

2019 ◽  
Vol 25 ◽  
pp. 260-265
Author(s):  
E. V. Lagunovskaya ◽  
O. I. Zaitseva ◽  
V. A. Lemesh
Keyword(s):  
Culture Medium ◽  
Medium Composition ◽  
Induction Medium ◽  
Hexaploid Triticale ◽  
Culture Methods ◽  
Study Results ◽  
Short Period ◽  
Androgenesis In Vitro ◽  
The Republic

Aim. Triticale is one of the main grain crops of the Republic of Belarus. Further progress in the selection of this culture involves the accelerated creation of highly productive early ripening varieties resistant to abiotic and biotic factors. The method of induced androgenesis in vitro makes it possible to obtain stable homozygous lines in a short period of time and to eliminate the lengthy process of inbreeding used in classical breeding to fix the desired traits. Methods. The tissue and cell culture methods for plants was used in the study. Results. The influence of the induction medium composition on the efficiency of in vitro induced androgenesis in varieties and lines of hexaploid triticale is assessed. The influence of three types of induction culture medium, the type of phytohormones and the presence or absence of cefotaxime in the medium are analyzed. Results. It has been shown that using the C-17 culture medium supplemented with 2.0 mg/l 2,4-D and 0.5 mg/l kinetin without adding cefotaxime is most effective for the anther triticale cultivation. Keywords: triticale, anther culture, induction nutrient medium, embryoids, calli, regenerant plants, cefotaxime.

scholarly journals Co-expression of glutathione S-transferase with methionine aminopeptidase: a system of producing enriched N-terminal processed proteins in Escherichia coli

1999 ◽  
Vol 338 (2) ◽  
pp. 335-342 ◽  
Author(s):  
Debbie D. W. HWANG ◽  
Li-Fan LIU ◽  
I-Ching KUAN ◽  
Lih-Yuan LIN ◽  
Tsuey-Chyi S. TAM ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Expression System ◽  
Methionine Aminopeptidase ◽  
Glutathione S Transferase ◽  
Methionine Residue ◽  
Methionine Residues ◽  
Processed Form ◽  
Escherichia Coli Expression ◽  
Rat Livers

We describe here an Escherichia coli expression system that produces recombinant proteins enriched in the N-terminal processed form, by using glutathione S-transferase cGSTM1-1 and rGSTT1-1 as models, where c and r refer to chick and rat respectively. Approximately 90% of the cGSTM1-1 or rGSTT1-1 overexpressed in E. coliunder the control of a phoA promoter retained the initiator methionine residue that was absent from the mature isoenzymes isolated from tissues. The amount of initiator methionine was decreased to 40% of the expressed cGSTM1-1 when the isoenzyme was co-expressed with an exogenous methionine aminopeptidase gene under the control of a separate phoA promoter. The recombinant proteins expressed were mainly methionine aminopeptidase. The yield of cGSTM1-1 was decreased to 10% of that expressed in the absence of the exogenous methionine aminopeptidase gene. By replacing the phoA with its natural promoter, the expression of methionine aminopeptidase decreased drastically. The yield of the co-expressed cGSTM1-1 was approx. 60% of that in the absence of the exogenous methionine aminopeptidase gene; approx. 65% of the initiator methionine residues were removed from the enzyme. Under similar conditions, N-terminal processing was observed in approx. 70% of the recombinant rGSTT1-1 expressed. By increasing the concentration of phosphate in the growth medium, the amount of initiator methionine on cGSTM1-1 was decreased to 14% of the overexpressed isoenzymes, whereas no further improvement could be observed for rGSTT1-1. The initiator methionine residue does not affect the enzymic activities of either cGSTM1-1 or rGSTT1-1. However, the epoxidase activity and the 4-nitrobenzyl chloride-conjugating activity of the purified recombinant rGSTT1-1 are markedly higher that those reported recently for the same isoenzyme isolated from rat livers.

scholarly journals The Effects of Temperature and Culture Medium Composition on Protein Recombinant JSU-pET Expression

2019 ◽  
Vol 17 (2) ◽  
pp. 31
Author(s):  
Indriawati Indriawati ◽  
Slamet Diah Volkandari ◽  
Endang Tri Margawati
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Culture Medium ◽  
Medium Composition ◽  
Protein Yield ◽  
Culture Temperature ◽  
Factors Influencing ◽  
Effects Of Temperature ◽  
The Right ◽  
Medium Culture

There are several factors influencing the protein expression. The study was aimed to increase the yield of protein recombinant JSU-pET by applying combination of temperature and medium culture composition. This research was composed in a 2x2 factorial design with two level temperatures of 37<sup>o</sup>C and 25<sup>o</sup>C and 2 medium compositions of LB and mLB (modified). <em>Escherichia coli</em> bearing JSU-pET plasmid were grown in two applied media and incubated in two applied temperatures. The cells were harvested by centrifugation the pellets were then lysed. The lysed cells were centrifuged, the supernatant then purified by Ni-NTA Resin Agarose. The purified JSU protein recombinant was characterized and quantified by spectrophotometer. The Result showed that the JSU protein recombinant was successfully expressed on the right size (37kDa) in all treatments even though the JSU-pET showed sharper band in m LB at 37<sup>o</sup>C. Statistically the protein yield was not significant different (P&gt;0.05), however culture temperature of 25<sup>o</sup>C showed higher yield (0.618±0.095 vs 0.704±0.094) than cultured at 37<sup>o</sup>C (0.598±0.137 vs 0.553±0.041), respectively either cultured in LB or mLB. This finding suggests that a lower culture temperature could increase protein yield both either in LB or modified LB.

Pertumbuhan Dan Perkembangan Anggrek Dendrobium anosmum Pada Media Kultur In Vitro Dengan Beberapa Konsentrasi Air Kelapa

Agrologia ◽  
2018 ◽  
Vol 1 (1) ◽  
Author(s):  
S. Tuhuteru ◽  
Meity L Hehanussa ◽  
Simon H.T Raharjo
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Water Concentration ◽  
Medium Composition ◽  
Coconut Water ◽  
Orchid Species ◽  
Randomized Design ◽  
Wet Weight ◽  
Completely Randomized Design

Dendrobium anosmum is one of natural orchids in Indonesia. Optimization of medium composition for orchid propagation through in vitro culture is necessary to enhance propagule multiplication capabilities and quality. This study was aimed to study the influence of concentration of coconut water in culture medium on in vitro growth and development of D. anosmum orchid species and to determine the optimal coconut water concentration in culture media.  The experiment were arranged in a Completely Randomized Design with four treatments and eight replications. The treatments consisted of the addition of coconut water with concentrations: 0 ml•l -1 (control), 50 ml•l-1, 100 ml•l-1 and 150 ml•l-1. The results showed that addition of coconut water in culture medium gave different effect on shoot growth and multiplication of D. anosmum orchids.  Coconut water concentration of 100 ml•l-1 was the best concentration for growth and multiplication of D. anosmum orchids, based on both shoots and roots growth, plantlet height and wet weight.

Differentiation of mesenchymal stem cells from humans and animals into insulin-producing cells: An overview in vitro induction forms

2020 ◽  
Vol 16 ◽  
Author(s):  
Bruna O. S. Câmara ◽  
Bruno M. Bertassoli ◽  
Natália M. Ocarino ◽  
Rogéria Serakides
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture Medium ◽  
Adipogenic Differentiation ◽  
Medium Composition ◽  
Cell Therapies ◽  
Insulin Producing Cells ◽  
Msc Differentiation

The use of stem cells in cell therapies has shown promising results in the treatment of several diseases, including diabetes mellitus, in both humans and animals. Mesenchymal stem cells (MSCs) can be isolated from various locations, including bone marrow, adipose tissues, synovia, muscles, dental pulp, umbilical cords, and the placenta. In vitro, by manipulating the composition of the culture medium or transfection, MSCs can differentiate into several cell lineages, including insulin-producing cells (IPCs). Unlike osteogenic, chondrogenic, and adipogenic differentiation, for which the culture medium and time are similar between studies, studies involving the induction of MSC differentiation in IPCs differ greatly. This divergence is usually evident in relation to the differentiation technique used, the composition of the culture medium, the cultivation time, which can vary from a few hours to several months, and the number of steps to complete differentiation. However, although there is no “gold standard” differentiation medium composition, most prominent studies mention the use of nicotinamide, exedin-4, ß-mercaptoethanol, fibroblast growth factor b (FGFb), and glucose in the culture medium to promote the differentiation of MSCs into IPCs. Therefore, the purpose of this review is to investigate the stages of MSC differentiation into IPCs both in vivo and in vitro, as well as address differentiation techniques and molecular actions and mechanisms by which some substances, such as nicotinamide, exedin-4, ßmercaptoethanol, FGFb, and glucose, participate in the differentiation process.

scholarly journals Lac Operon Boolean Models: Dynamical Robustness and Alternative Improvements

Mathematics ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 600 ◽  
Author(s):  
Marco Montalva-Medel ◽  
Thomas Ledger ◽  
Gonzalo A. Ruz ◽  
Eric Goles
Keyword(s):  
Escherichia Coli ◽  
Limit Cycles ◽  
The Other ◽  
Lac Operon ◽  
Boolean Models ◽  
Bistable Behavior

In Veliz-Cuba and Stigler 2011, Boolean models were proposed for the lac operon in Escherichia coli capable of reproducing the operon being OFF, ON and bistable for three (low, medium and high) and two (low and high) parameters, representing the concentration ranges of lactose and glucose, respectively. Of these 6 possible combinations of parameters, 5 produce results that match with the biological experiments of Ozbudak et al., 2004. In the remaining one, the models predict the operon being OFF while biological experiments show a bistable behavior. In this paper, we first explore the robustness of two such models in the sense of how much its attractors change against any deterministic update schedule. We prove mathematically that, in cases where there is no bistability, all the dynamics in both models lack limit cycles while, when bistability appears, one model presents 30% of its dynamics with limit cycles while the other only 23%. Secondly, we propose two alternative improvements consisting of biologically supported modifications; one in which both models match with Ozbudak et al., 2004 in all 6 combinations of parameters and, the other one, where we increase the number of parameters to 9, matching in all these cases with the biological experiments of Ozbudak et al., 2004.

Sign in / Sign up

Export Citation Format

Share Document