Lactose-induced Production of Human Soluble B Lymphocyte Stimulator (hsBLyS) in E. coli with Different Culture Strategies

Li, Zhaopeng; Zhang, Xu; Tan, Tianwei

doi:10.1007/s10529-006-0002-y

Cited by 14 publications

(8 citation statements)

References 12 publications

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“…The lactose concentration of 20 g.L -1 in the moment of induction was superior to values commonly reported in the literature (Li et al 2006; Neubauer et al 1992). It led to an amount of 76.5 g of lactose totally consumed within 3.3 h after its addition.…”

Section: Resultscontrasting

confidence: 60%

“…coli BL21(DE3) fed-batch cultures in complex medium Product & molecular weightCarbon source/inducerCulture time (h)Biomass productivity (g DCW .L -1 .h -1 )Protein volumetric productivity (g prot . L -1 .h -1 )Specific protein production (g prot .g DCW -1 )Product content (g.L -1 )ReferenceHuman soluble B lymphocyte stimulator (hsBLyS) 17-18 kDaGlucose/lactose340.920.110.1203.70Li et al 2006Tilapia insulin-like growth factor 2 - 7 kDaGlucose/IPTG305.200.320.0629.69Hu et al 2004Streptokinase 47 kDaGlucose/IPTG111.310.100.0501.12Goyal et al 2009Anti-cancer drug TATm-survivin (T34A) 17 kDaGlucose/IPTG140.630.120.1911.68Zhang et al 2009 (t = 14 h)Anti-HIV protein Griffthsin (GRFT) 14.5 kDaAuto induction (Glucose + glycerol + lactose)/lactose150.89 a 0.050.061 a 0.82Giomarelli et al 2006 this work: SpaA from E . rhusiopathiae 42 kDaGlucose/IPTG11.51.820.250.1362.85Exp #1 (t = 11.5 h)Auto induction (Glucose + glycerol + lactose)/lactose201.160.18…”

Section: Resultsmentioning

confidence: 99%

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Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures

et al. 2013

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In spite of the large number of reports on fed-batch cultivation of E. coli, alternative cultivation/induction strategies remain to be more deeply exploited. Among these strategies, it could be mentioned the use of complex media with combination of different carbon sources, novel induction procedures and feed flow rate control matching the actual cell growth rate. Here, four different carbon source combinations (glucose, glycerol, glucose + glycerol and auto-induction) in batch media formulation were compared. A balanced combination of glucose and glycerol in a complex medium formulation led to: fast growth in the batch-phase; reduced plasmid instability by preventing early expression leakage; and protein volumetric productivity of 0.40 g.L-1.h-1. Alternative induction strategies were also investigated. A mixture of lactose and glycerol as supplementary medium fully induced a high biomass population, reaching a good balance between specific protein production (0.148 gprot.gDCW-1) and volumetric productivity (0.32 g.L-1.h-1). The auto-induction protocol showed excellent results on specific protein production (0.158 gprot.gDCW-1) in simple batch cultivations. An automated feed control based on the on-line estimated growth rate was implemented, which allowed cells to grow at higher rates than those generally used to avoid metabolic overflow, without leading to acetate accumulation. Some of the protocols described here may provide a useful alternative to standard cultivation and recombinant protein production processes, depending on the performance index that is expected to be optimized. The protocols using glycerol as carbon source and induction by lactose feeding, or glycerol plus glucose in batch medium and induction by lactose pulse led to rSpaA production in the range of 6 g.L-1, in short fed-batch processes (16 to 20 h) with low accumulation of undesired side metabolites.

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Section: Resultscontrasting

confidence: 60%

Section: Resultsmentioning

confidence: 99%

Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures

et al. 2013

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“…We used glycerol instead of glucose as main carbon- and energy source to avoid interferences with the glucose sensitive P BAD promoter. In contrast to glucose, levels of cAMP are high and acetate accumulation is low even if excess amounts of glycerol are present in E. coli cultures [32,33]. …”

Section: Resultsmentioning

confidence: 99%

Production of glycoprotein vaccines in Escherichia coli

Kowarik²,

et al. 2010

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BackgroundConjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries.ResultsTwo different periplasmic carrier proteins, AcrA from C. jejuni and a toxoid form of Pseudomonas aeruginosa exotoxin were glycosylated with Shigella O antigens in E. coli. Starting from shake flask cultivation in standard complex medium a lab-scale fed-batch process was developed for glycoconjugate production. It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction. After induction glycoconjugates generally appeared later than unglycosylated carrier protein, suggesting that glycosylation was the rate-limiting step for synthesis of conjugate vaccines in E. coli. Glycoconjugate synthesis, in particular expression of oligosaccharyltransferase PglB, strongly inhibited growth of E. coli cells after induction, making it necessary to separate biomass growth and recombinant protein expression phases. With a simple pulse and linear feed strategy and the use of semi-defined glycerol medium, volumetric glycoconjugate yield was increased 30 to 50-fold.ConclusionsThe presented data demonstrate that glycosylated proteins can be produced in recombinant E. coli at a larger scale. The described methodologies constitute an important step towards cost-effective in vivo production of conjugate vaccines, which in future may be used for combating severe infectious diseases, particularly in developing countries.

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“…The strict requirement for oxygen-limited conditions during induction hinders a scale-up to higher biomass concentrations. Oxygen saturation in high cell density fed-batch cultures of E. coli is usually kept above 10% in order to avoid a shift to fermentative growth3637, as this would lead to rapid accumulation of toxic concentrations of organic acids. Induction of LMCO expression and shift to oxygen limitation very late in the process may solve the problem.…”

Section: Discussionmentioning

confidence: 99%

Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli

Ihssen

Reiss

Luchsinger

et al. 2015

Sci Rep

105

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Laccases are multi-copper oxidases that oxidize a broad range of substrates at the expense of molecular oxygen, without any need for co-factor regeneration. These enzymes bear high potential for the sustainable synthesis of fine chemicals and the modification of (bio)polymers. Here we describe cloning and expression of five novel bacterial laccase-like multi copper oxidases (LMCOs) of diverse origin which were identified by homology searches in online databases. Activity yields under different expression conditions and temperature stabilities were compared to three previously described enzymes from Bacillus subtilis, Bacillus pumilus and Bacillus clausii. In almost all cases, a switch to oxygen-limited growth conditions after induction increased volumetric activity considerably. For proteins with predicted signal peptides for secretion, recombinant expression with and without signal sequence was investigated. Bacillus CotA-type LMCOs outperformed enzymes from Streptomyces and Gram-negative bacteria with respect to activity yields in Escherichia coli and application relevant biochemical properties. The novel Bacillus coagulans LMCO combined high activity yields in E. coli with unprecedented activity at strong alkaline pH and high storage stability, making it a promising candidate for further development.

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Lactose-induced Production of Human Soluble B Lymphocyte Stimulator (hsBLyS) in E. coli with Different Culture Strategies

Cited by 14 publications

References 12 publications

Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures

Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures

Production of glycoprotein vaccines in Escherichia coli

Biochemical properties and yields of diverse bacterial laccase-like multicopper oxidases expressed in Escherichia coli

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