Efficient Production of a Thermophilic 2-Deoxyribose-5-Phosphate Aldolase in Glucose-Limited Fed-Batch Cultivations of Escherichia coli by Continuous Lactose Induction Strategy

Pei, Xiaolin; Wang, Qiuyan; Li, Chenglu; Qiu, Xiaoqing; Xie, Kailin; Huang, Lifeng; Wang, Anming; Zeng, Zhigang; Xie, Tian

doi:10.1007/s12010-011-9261-8

Cited by 13 publications

(4 citation statements)

References 23 publications

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“…In this study we investigated the effects of lactose induction on an E. coli BL21 (DE3) expression system with a genome-integrated GOI. 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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“…Thus, lactose has been studied as alternative inducer. Lactose was found to be as effective as IPTG, to increase cell fitness, to reduce IB formation, and to enhance the formation of soluble recombinant product (Bashir et al 2015 ; Fruchtl et al 2015 ; Gombert and Kilikian 1998 ; Neubauer et al 1992 ; Pei et al 2011 ; Zou et al 2014 ). However, lactose is metabolized by E. coli making stable induction more complicated as it has to be continuously supplied (Striedner et al 2003 ).…”

Section: Introductionmentioning

confidence: 99%

The E. coli pET expression system revisited—mechanistic correlation between glucose and lactose uptake

Wurm

Veiter

Ulonska

et al. 2016

Appl Microbiol Biotechnol

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Therapeutic monoclonal antibodies are mainly produced in mammalian cells to date. However, unglycosylated antibody fragments can also be produced in the bacterium Escherichia coli which brings several advantages, like growth on cheap media and high productivity. One of the most popular E. coli strains for recombinant protein production is E. coli BL21(DE3) which is usually used in combination with the pET expression system. However, it is well known that induction by isopropyl β-d-1-thiogalactopyranoside (IPTG) stresses the cells and can lead to the formation of insoluble inclusion bodies. In this study, we revisited the pET expression system for the production of a novel antibody single-chain variable fragment (scFv) with the goal of maximizing the amount of soluble product. Thus, we (1) investigated whether lactose favors the recombinant production of soluble scFv compared to IPTG, (2) investigated whether the formation of soluble product can be influenced by the specific glucose uptake rate (qs,glu) during lactose induction, and (3) determined the mechanistic correlation between the specific lactose uptake rate (qs,lac) and qs,glu. We found that lactose induction gave a much greater amount of soluble scFv compared to IPTG, even when the growth rate was increased. Furthermore, we showed that the production of soluble protein could be tuned by varying qs,glu during lactose induction. Finally, we established a simple model describing the mechanistic correlation between qs,lac and qs,glu allowing tailored feeding and prevention of sugar accumulation. We believe that this mechanistic model might serve as platform knowledge for E. coli.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7620-7) contains supplementary material, which is available to authorized users.

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“…The culture medium and system with the best protein yield per volume was TB in the bioreactor (approximately 10 mg/L); the 2x (YT) medium in the bioreactor yielded almost half that value (5 mg/L of recombinant protein; Fig 4C ). The higher growth yields observed in the fermenter can be ascribed to greater control of the aerobic culture conditions, which contributed more energy for protein synthesis [ 33 ].…”

Section: Resultsmentioning

confidence: 99%

Molecular characterization of the recombinant protein RmLTI-BmCG-LTB: Protective immunity against Rhipicephalus (Boophilus) microplus

et al. 2018

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The bovine tick Rhipicephalus (Boophilus) microplus is found in several tropical and subtropical regions of the world. This parasite transmits pathogens that cause disease, such as babesiosis (Babesia bovis and B. bigemina) and anaplasmosis (Anaplasma marginale). Tick infestations cause enormous livestock losses, and controlling tick infestations and the transmission of tick-borne diseases remains a challenge for the livestock industry. Because the currently available commercial vaccines offer only partial protection against R. (B.) microplus, there is a need for more efficient vaccines. Several recombinant antigens have been evaluated using different immunization strategies, and they show great promise. This work describes the construction and immunological characterization of a multi-antigen chimera composed of two R. (B.) microplus antigens (RmLTI and BmCG) and one Escherichia coli antigen (B subunit, LTB). The immunogenic regions of each antigen were selected and combined to encode a single polypeptide. The gene was cloned and expressed in E. coli. For all of the experiments, two groups (treated and control) of four Angus heifers (3–6 months old) were used. The inoculation was performed via intramuscular injection with 200 μg of purified recombinant chimeric protein and adjuvated. The chimeric protein was recognized by specific antibodies against each subunit and by sera from cattle inoculated with the chimera. Immunization of RmLTI-BmCG-LTB cattle reduced the number of adult female ticks by 6.29% and vaccination of cattle with the chimeric antigen provided 55.6% efficacy against R. (B.) microplus infestation. The results of this study indicate that the novel chimeric protein is a potential candidate for the future development of a more effective vaccine against R. (B.) microplus.

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Efficient Production of a Thermophilic 2-Deoxyribose-5-Phosphate Aldolase in Glucose-Limited Fed-Batch Cultivations of Escherichia coli by Continuous Lactose Induction Strategy

Cited by 13 publications

References 23 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

The E. coli pET expression system revisited—mechanistic correlation between glucose and lactose uptake

Molecular characterization of the recombinant protein RmLTI-BmCG-LTB: Protective immunity against Rhipicephalus (Boophilus) microplus

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