Lactose Induction Increases Production of Recombinant Keratinocyte Growth Factor-2 in Escherichia coli

Tian, Haishan; Tang, Lu; Wang, Yi; Wang, Xiaojie; Guan, Lili; Zhang, Jian; Wu, Xiaoping; Li, Xiaokun

doi:10.1007/s10989-011-9249-9

Cited by 14 publications

(13 citation statements)

References 17 publications

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“…Previously, only plasmid-based expression systems had been subject to studies investigating lactose as an inducer for recombinant protein production [23,36,37]. In those studies, beneficial effects on productivity, cell fitness, viability, and soluble product formation had been reported [21,22,[38][39][40].…”

Section: Discussionmentioning

confidence: 99%

The Effects of Lactose Induction on a Plasmid-Free E. coli T7 Expression System

et al. 2020

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Recombinant production of pharmaceutical proteins like antigen binding fragments (Fabs) in the commonly-used production host Escherichia coli presents several challenges. The predominantly-used plasmid-based expression systems exhibit the drawback of either excessive plasmid amplification or plasmid loss over prolonged cultivations. To improve production, efforts are made to establish plasmid-free expression, ensuring more stable process conditions. Another strategy to stabilize production processes is lactose induction, leading to increased soluble product formation and cell fitness, as shown in several studies performed with plasmid-based expression systems. Within this study we wanted to investigate lactose induction for a strain with a genome-integrated gene of interest for the first time. We found unusually high specific lactose uptake rates, which we could attribute to the low levels of lac-repressor protein that is usually encoded not only on the genome but additionally on pET plasmids. We further show that these unusually high lactose uptake rates are toxic to the cells, leading to increased cell leakiness and lysis. Finally, we demonstrate that in contrast to plasmid-based T7 expression systems, IPTG induction is beneficial for genome-integrated T7 expression systems concerning cell fitness and productivity.

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Section: Discussionmentioning

confidence: 99%

The Effects of Lactose Induction on a Plasmid-Free E. coli T7 Expression System

et al. 2020

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“…In previous studies, the time chosen for induction was when the OD 600nm was between 0.40 and 0.80 [ 38 , 47 , 51 , 52 , 53 ]. Since IPTG is toxic to cells, if added at the beginning of the cultivation there will be a loss in the final biomass concentration [ 27 ]. The interval of best L-ASNase activity was the same for both inducers used with no statistical difference between them.…”

Section: Resultsmentioning

confidence: 99%

“…To determine the best concentration of each inductor, three concentrations were chosen based on previous studies. For IPTG, concentration often ranges from 0.10 mmol L −1 to 1.00 mmol L −1 [ 21 , 22 , 23 , 24 ] while lactose concentration varies from 10 g L −1 to 18 g L −1 [ 25 , 26 , 27 ]. Considering the reported concentrations, IPTG was evaluated at 0.10 mmol L −1 , 0.45 mmol L −1 and 1.00 mmol L −1 and lactose was evaluated at 10 g L −1 , 14 g L −1 and 18 g L −1 .…”

Section: Methodsmentioning

confidence: 99%

Development of Processes for Recombinant L-Asparaginase II Production by Escherichia coli Bl21 (De3): From Shaker to Bioreactors

et al. 2020

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Since 1961, L-asparaginase has been used to treat patients with acute lymphocytic leukemia. It rapidly depletes the plasma asparagine and deprives the blood cells of this circulating amino acid, essential for the metabolic cycles of cells. In the search for viable alternatives to produce L-asparaginase, this work aimed to produce this enzyme from Escherichia coli in a shaker and in a 3 L bioreactor. Three culture media were tested: defined, semi-defined and complex medium. L-asparaginase activity was quantified using the β-hydroxamate aspartic acid method. The defined medium provided the highest L-asparaginase activity. In induction studies, two inducers, lactose and its analog IPTG, were compared. Lactose was chosen as an inducer for the experiments conducted in the bioreactor due to its natural source, lower cost and lower toxicity. Batch and fed-batch cultures were carried out to reach high cell density and then start the induction. Batch cultivation provided a final cell concentration of 11 g L−1 and fed-batch cultivation produced 69.90 g L−1 of cells, which produced a volumetric activity of 43,954.79 U L−1 after lactose induction. L-asparaginase was produced in a shaker and scaled up to a bioreactor, increasing 23-fold the cell concentration and thus, the enzyme productivity.

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“…Some of the most used techniques in biotechnology involves protein expression induction by pumping an inductor (e.g. Isopropyl β-D-1-thiogalactopyranoside -IPTG) or lactose (Lin & Hsu, 1997;Tian et al, 2011;Faust, Stand & Weuster-Botz, 2015). For maximum yield of product of interest, the induction is optimized to take place when the culture has reached a specified density (Glazyrina et al, 2010).…”

Section: Protein Expressionmentioning

confidence: 99%

“…Tight control of pH, the induction time of protein expression and temperature contribute to a much higher biomass and protein yield in biotechnological process (Gueguim et al, 2003). The precise control of the protein expression induction is critical for maximizing the useful product yield (Tian et al, 2011). Inducing a culture too early, may prevent its growth, while inducing it too late may not have enough nutrients left in the fermentation broth to support the biosynthesis of the desired product.…”

Section: Protein Expression and Culture Harvest Timementioning

confidence: 99%

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Supplemental Information 6: Supplement 6

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Growing microorganisms for laboratory experiments or industrial biotechnological process is an activity which involves the use of bioreactors. Although there are many commercially available equipment, most of them lack the flexibility of an open-source solution. This work proposes a cost effective Arduino based bioreactor controller for growing suspended microbial cells. To exemplify its functionality, this study provides the parts list and schematics necessary to make a functional laboratory scale bench top stirred tank bioreactor. Using the built prototype, an E. coli culture is grown maintaining the preset parameters, protein expression is induced and culture is harvested at preset culture density. Automatically recorded process data shows stable environmental parameters and reliable bacterial growing curve.

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Lactose Induction Increases Production of Recombinant Keratinocyte Growth Factor-2 in Escherichia coli

Cited by 14 publications

References 17 publications

The Effects of Lactose Induction on a Plasmid-Free E. coli T7 Expression System

The Effects of Lactose Induction on a Plasmid-Free E. coli T7 Expression System

Development of Processes for Recombinant L-Asparaginase II Production by Escherichia coli Bl21 (De3): From Shaker to Bioreactors

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