Recombinant gene expression in Escherichia coli cultivation using lactose as inducer

Gombert, Andreas; Kilikian, Beatriz Vahan

doi:10.1016/s0168-1656(97)00185-5

Cited by 66 publications

(48 citation statements)

References 16 publications

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“…The plasmid replication control mechanism can be restored by eliminating sequence homologies between ColE1 RNA I/RNA II and tRNAs; however, thusly modified pET systems with a stabilized plasmid copy number remain too strong (Grabherr et al, 2002). Processing strategies developed to improve control of the expression rate, such as the transcription tuning concept (Striedner et al, 2003) or the application of lactose as an inducer with lower affinity for the operator binding site (Gombert and Kilikian, 1998;Lin and Hsu, 1997;Studier, 2005), have been partially successful, but the challenge of designing a comprehensive solution for controlling the production scale remains unmet.…”

Section: Introductionmentioning

confidence: 99%

Plasmid‐free T7‐based Escherichia coli expression systems

Striedner

Pfaffenzeller

Luchner

et al. 2010

Biotech & Bioengineering

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In order to release host cells from plasmid-mediated increases in metabolic load and high gene dosages, we developed a plasmid-free, T7-based E. coli expression system in which the target gene is site-specifically integrated into the genome of the host. With this system, plasmid-loss, a source of instability for conventional expression systems, was eliminated. At the same time, system leakiness, a challenging problem with recombinant systems, was minimized. The efficiency of the T7 RNA polymerase compensates for low gene dosage and provides high rates of recombinant gene expression without fatal consequences to host metabolism. Relative to conventional pET systems, this system permits improved process stability and increases the host cell's capacity for recombinant gene expression, resulting in higher product yields. The stability of the plasmid-free system was proven in chemostat cultivation for 40 generations in a non-induced and for 10 generations in a fully induced state. For this reason plasmid-free systems benefit the development of continuous production processes with E. coli. However, time and effort of the more complex cloning procedure have to be considered in relation to the advantages of plasmid-free systems in upstream-processing.

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Section: Introductionmentioning

confidence: 99%

Plasmid‐free T7‐based Escherichia coli expression systems

Striedner

Pfaffenzeller

Luchner

et al. 2010

Biotech & Bioengineering

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“…The IPTG inducible promoters lac, tac, and trc (Makrides, 1996) are powerful and widely used for basic research. However, the use of IPTG for the large-scale production of recombi-nant proteins is undesirable because of its high cost and toxicity (Donovan et al, 1996;Figge et al, 1988;Gombert and Kilikian, 1998;Kosinski et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

“…In Lac + host strains the promoters mentioned above can also be induced by the addition of lactose, which has the double role of inducer and carbon and energy source (Donovan et al, 1996;Gombert and Kilikian, 1998;Kapralek et al, 1991;Kilikian et al, 2000;Neubauer et al, 1991;Neubauer and Hofmann, 1994;Woyski and Cupp-Vickery, 2001). Despite its low cost, the use of lactose as inducer has several drawbacks, which results in low volumetric yields and difficulties in process control (Gombert and Kilikian, 1998).…”

Section: Introductionmentioning

confidence: 99%

Novel escherichia coli strain allows efficient recombinant protein production using lactose as inducer

Menzella¹,

Ceccarelli²,

Gramajo³

2003

Biotech & Bioengineering

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An important characteristic of promoters used in recombinant protein production in Escherichi coli is their inducibility in a simple and cost-effective manner. The IPTG inducible promoters lac, tac, and trc are powerful and widely used for basic research. However, the use of IPTG in large-scale production is undesirable due to its high cost and toxicity. The promoters mentioned above can also be induced by the addition of lactose, which has the double role of inducer and carbon and energy source. Nevertheless, the use of this sugar in industrial processes has several drawbacks, which result in low volumetric yields and difficulties in process control. We have genetically engineered a BL21 strain to allow the efficient use of lactose as inducer in fed-batch cultures. Two modifications were introduced, the exchange of the wild-type lac operator by a constitutive one (lacO(c)) and the replacement of the gal alleles to recover the Gal(+) phenotype. The constitutive expression of the lac operon overcame the negative effects that the Lac nongenetic heterogeneity of wild-type E. coli introduces when lactose is used as inducer. The gal(+) genotype allowed the complete use of the lactose as carbon and energy source. The relevance of these two modifications in the efficient utilization of lactose as inducer was demonstrated in fed-batch cultures of the novel recombinant strain MP101 harboring expression vectors containing the calf prochymosin gene or the pccB gene, which encodes for the carboxyltransferase component of a propionyl-CoA carboxylase complex from Streptomyces coelicolor. Similar levels of recombinant protein production (up to 16 g/L) were obtained by using either lactose or IPTG as inducers, which confirmed the success of the genetics modifications introduced.

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“…Os cultivos são realizados em meios definidos ou semi-definidos, com concentrações dos substratos inferiores às inibitórias e especialmente controlando a concentração da fonte de carbono 88 . Os cultivos são, em geral, descontínuos alimentados, tendo diversas estratégias na fase de alimentação.…”

Section: Princípios De Cultivo Para Ampliação De Escalaunclassified

“…Alternativamente, pode-se utilizar para a indução, a própria lactose, onde esta pode apresentar um duplo papel, ser usada tanto como um indutor quanto fonte de energia 75,76 . Porém, o uso deste açúcar em processos industriais pode apresentar obstáculos, já que às vezes resulta em baixo rendimento volumétrico e dificuldades no controle do processo 77,78 .…”

Section: Introdução -39 -unclassified

Clonagem, expressão e purificação das proteínas de superfície, PsaA e fragmentos de PspA de Streptococcus pneumoniae

Silva¹

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Recombinant gene expression in Escherichia coli cultivation using lactose as inducer

Cited by 66 publications

References 16 publications

Plasmid‐free T7‐based Escherichia coli expression systems

Plasmid‐free T7‐based Escherichia coli expression systems

Novel escherichia coli strain allows efficient recombinant protein production using lactose as inducer

Clonagem, expressão e purificação das proteínas de superfície, PsaA e fragmentos de PspA de Streptococcus pneumoniae

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