Control of misincorporation of de novo synthesized norleucine into recombinant interleukin-2 in E. coli

Tsai, Larry B.; Lu, Hsieng S.; Kenney, William C.; Curless, Craig C.; Klein, Michael L.; Lai, Por-Hsiung; Fenton, Dennis; Altrock, Bruce W.; Mann, Michael B.

doi:10.1016/s0006-291x(88)80904-5

Cited by 50 publications

(22 citation statements)

References 5 publications

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“…The incorporation of modifi ed amino acids in recombinant proteins has been assigned to (i) conditions under which the branched -chain amino acid pathway gets derepressed, such as strong expression of leucine -rich proteins [55] that cause an exhaustion of the proteinogenic amino acid (e.g., leucine), (ii) in combination with an expression in mineral salt media, and (iii) a high (external) concentration of these amino acids.…”

Section: Effects Of Conditions In Industrial -Scale Fed -Batch Procesmentioning

confidence: 99%

Consistency of Scale‐Up from Bioprocess Development to Production

Junne

Klingner

Itzeck

et al. 2012

Biopharmaceutical Production Technology

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The fed -batch technique is the most frequently applied operation mode for an effi cient biotechnological production. It is often characterized by the achievement of higher product yields compared to the batch mode. For the case when the fedbatch process is operated under substrate -limiting conditions, substrate uptake rates lower than the maximum uptake capacities of cells occur. Thus, the accumulation of branch -point intermediate compounds of the cell ' s metabolism is more or less avoided. The synthesis of unwanted side -products is not at all or only supported barely with low amounts of substrate. Usually, under substrate -limiting conditions, carbon is provided for the most important products in order to keep the cellular system functional and viable.Such substrate -limiting conditions are easily achieved at the laboratory scale and pilot scale. Most often, the feed solution is introduced through a pipe at the top of the fermenter. A mixing time of several seconds is achieved in stirred tank reactor s ( STR s), which is so fast that the even distribution of the substrate in the reactor is not disturbed. Hence, even up to high cell densities, no accumulation of unwanted byproducts under fed -batch conditions is observed, except that viscosity is very high and oxygen availability becomes insuffi cient in late process stages. However, this is different in biotechnological production in large -scale fermenters. Due to mechanical and economical limitations of the power input, the mixing time of an STR increases 10 -fold or more when reaching liquid volumes of several cubic meters [1 -3] . This leads to a faster conversion by the microorganisms near the feeding zone, while far away from it production of the substrate ceases with increasing cell concentration. The resulting gradients near the feeding point alter the process performance [4,5] . In particular, substrate excess, which thwarts the idea of a fed -batch process, can lead to the synthesis of unwanted byproducts [6] . Due to the movement of cells in the reactor, they are exposed to ongoing oscillating environmental conditions where they fl ip between substrate excess and 16 Biopharmaceutical Production Technology, First Edition. Edited by Ganapathy Subramanian. Glucose metabolism at high density growth of E. coli B and E. coli K: differences in metabolic pathways are

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Section: Effects Of Conditions In Industrial -Scale Fed -Batch Procesmentioning

confidence: 99%

Consistency of Scale‐Up from Bioprocess Development to Production

Junne

Klingner

Itzeck

et al. 2012

Biopharmaceutical Production Technology

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“…Interestingly, in the same study, it was discovered that ∼6% of the methionine residues in native E. coli proteins were also substituted by norleucine. Production of a therapeutically relevant protein, Interleukin‐2, in a minimal medium E. coli fermentation resulted in ∼19% of the methionine residues in the recombinant protein substituted with norleucine . Norleucine competes with methionine for incorporation into proteins due to the promiscuity of the methionyl tRNA synthetase (MetG) .…”

Section: Introductionmentioning

confidence: 99%

“…One method to reduce norleucine incorporation is to delete the genes involved in its biosynthetic pathway. Deleting the genes coding for leucine biosynthesis ( leuA , leuB , leuC , and leuD ) and/or the transaminases ( ilvE or tyrB ) have been successfully used to prevent norleucine incorporation . However, deleting the leucine biosynthetic pathway genes to prevent norleucine incorporation may require supplementation of high levels of leucine and other branched chain amino acids to the culture medium to support recombinant protein production.…”

Section: Introductionmentioning

confidence: 99%

“…Incorporation of norleucine at methionine positions during recombinant protein production in E. coli has been known for over 50 years. 3,5,6 For example, about 14% of methionine residues in methionyl bovine somatotropin (MBS) exhibited norleucine incorporation during production of this protein in E. coli. 3 Interestingly, in the same study, it was discovered that 6% of the methionine residues in native E. coli proteins were also substituted by norleucine.…”

Section: Introductionmentioning

confidence: 99%

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Strain engineering to prevent norleucine incorporation during recombinant protein production in Escherichia coli

Veeravalli¹,

Laird²,

Fedesco³

et al. 2014

Biotechnology Progress

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Incorporation of norleucine in place of methionine residues during recombinant protein production in Escherichia coli is well known. Continuous feeding of methionine is commonly used in E. coli recombinant protein production processes to prevent norleucine incorporation. Although this strategy is effective in preventing norleucine incorporation, there are several disadvantages associated with continuous feeding. Continuous feeding increases the operational complexity and the overall cost of the fermentation process. In addition, the continuous feed leads to undesirable dilution of the fermentation medium possibly resulting in lower cell densities and recombinant protein yields. In this work, the genomes of three E. coli hosts were engineered by introducing chromosomal mutations that result in methionine overproduction in the cell. The recombinant protein purified from the fermentations using the methionine overproducing hosts had no norleucine incorporation. Furthermore, these studies demonstrated that the fermentations using one of the methionine overproducing hosts exhibited comparable fermentation performance as the control host in three different recombinant protein production processes.

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“…Similarly, norleucine and norvaline have been shown to be synthesized as side-products of branched chain amino acid biosynthesis [2]. Norleucine is incorporated with alacrity into proteins, replacing up to 20% of methionine residues once methionine has been exhausted during protein overexpression [11,12]. …”

Section: Introductionmentioning

confidence: 99%

Evolution of phage with chemically ambiguous proteomes

2003

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Background: The widespread introduction of amino acid substitutions into organismal proteomes has occurred during natural evolution, but has been difficult to achieve by directed evolution. The adaptation of the translation apparatus represents one barrier, but the multiple mutations that may be required throughout a proteome in order to accommodate an alternative amino acid or analogue is an even more daunting problem. The evolution of a small bacteriophage proteome to accommodate an unnatural amino acid analogue can provide insights into the number and type of substitutions that individual proteins will require to retain functionality.

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Control of misincorporation of de novo synthesized norleucine into recombinant interleukin-2 in E. coli

Cited by 50 publications

References 5 publications

Consistency of Scale‐Up from Bioprocess Development to Production

Consistency of Scale‐Up from Bioprocess Development to Production

Strain engineering to prevent norleucine incorporation during recombinant protein production in Escherichia coli

Evolution of phage with chemically ambiguous proteomes

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