Isolation and Characterization of Acetylated Derivative of Recombinant Insulin Lispro Produced in Escherichia coli

Szewczak, Joanna; Bierczyńska-Krzysik, Anna; Piejko, Marcin; Mak, Paweł; Stadnik, Dorota

doi:10.1007/s11095-015-1637-y

Cited by 7 publications

(5 citation statements)

References 38 publications

(34 reference statements)

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“…Each acetylated isoform of a recombinant protein has the potential to have different biological implications. To reduce the number of acetylated isoforms, excess carbon should be avoided (21), and the growth medium should be buffered and supplemented with magnesium.…”

Section: Discussionmentioning

confidence: 99%

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

Christensen

Orr

Rao

et al. 2017

Appl Environ Microbiol

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Complex media are routinely used to cultivate diverse bacteria. However, this complexity can obscure the factors that govern cell growth. While studying protein acetylation in buffered tryptone broth supplemented with glucose (TB7-glucose), we observed that Escherichia coli did not fully consume glucose prior to stationary phase. However, when we supplemented this medium with magnesium, the glucose was completely consumed during exponential growth, with concomitant increases in cell number and biomass but reduced cell size. Similar results were observed with other sugars and other peptide-based media, including lysogeny broth. Magnesium also limited cell growth for Vibrio fischeri and Bacillus subtilis in TB7-glucose. Finally, magnesium supplementation reduced protein acetylation. Based on these results, we conclude that growth in peptide-based media is magnesium limited. We further conclude that magnesium supplementation can be used to tune protein acetylation without genetic manipulation. These results have the potential to reduce potentially deleterious acetylated isoforms of recombinant proteins without negatively affecting cell growth.IMPORTANCE Bacteria are often grown in complex media. These media are thought to provide the nutrients necessary to grow bacteria to high cell densities. In this work, we found that peptide-based media containing a sugar are magnesium limited for bacterial growth. In particular, magnesium supplementation is necessary for the bacteria to use the sugar for cell growth. Interestingly, in the absence of magnesium supplementation, the bacteria still consume the sugar. However, rather than use it for cell growth, the bacteria instead use the sugar to acetylate lysines on proteins. As lysine acetylation may alter the activity of proteins, this work demonstrates how lysine acetylation can be tuned through magnesium supplementation. These findings may be useful for recombinant protein production, when acetylated isoforms are to be avoided. They also demonstrate how to increase bacterial growth in complex media.KEYWORDS acetylation, complex media, improved cell growth, magnesium, tryptone B acteria are routinely grown in complex media containing a rich assortment of nutrients. For example, lysogeny broth (LB) contains tryptone and yeast exact. Together, these components provide a rich mixture of nutrients that supports growth of numerous bacteria. However, the complexity of such media obscures factors governing cell behavior, especially those that limit cell growth.We routinely grow Escherichia coli aerobically in buffered tryptone broth (TB7) supplemented with 4 g/liter glucose (TB7-glucose) when studying protein acetylation.

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Section: Discussionmentioning

confidence: 99%

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

Christensen

Orr

Rao

et al. 2017

Appl Environ Microbiol

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“…Finally, researchers should remember that heterologous expression of a protein in E. coli or any other bacterium carries the possibility that the purified protein preparation may be a heterogeneous mixture decorated by various acylations. Indeed, at least one group has reported that recombinant insulin produced in E. coli is acetylated (Szewczak et al, 2015). The potential for acylations to influence efficacy has great implications for the pharmaceutical industry, but also for any researcher who uses a heterologous system to express their protein of interest.…”

Section: Future Thoughtsmentioning

confidence: 99%

Post-translational Protein Acetylation: An Elegant Mechanism for Bacteria to Dynamically Regulate Metabolic Functions

et al. 2019

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Post-translational modifications (PTM) decorate proteins to provide functional heterogeneity to an existing proteome. The large number of known PTMs highlights the many ways that cells can modify their proteins to respond to diverse stimuli. Recently, PTMs have begun to receive increased interest because new sensitive proteomics workflows and structural methodologies now allow researchers to obtain large-scale, in-depth and unbiased information concerning PTM type and site localization. However, few PTMs have been extensively assessed for functional consequences, leaving a large knowledge gap concerning the inner workings of the cell. Here, we review understanding of N -𝜀-lysine acetylation in bacteria, a PTM that was largely ignored in bacteria until a decade ago. Acetylation is a modification that can dramatically change the function of a protein through alteration of its properties, including hydrophobicity, solubility, and surface properties, all of which may influence protein conformation and interactions with substrates, cofactors and other macromolecules. Most bacteria carry genes predicted to encode the lysine acetyltransferases and lysine deacetylases that add and remove acetylations, respectively. Many bacteria also exhibit acetylation activities that do not depend on an enzyme, but instead on direct transfer of acetyl groups from the central metabolites acetyl coenzyme A or acetyl phosphate. Regardless of mechanism, most central metabolic enzymes possess lysines that are acetylated in a regulated fashion and many of these regulated sites are conserved across the spectrum of bacterial phylogeny. The interconnectedness of acetylation and central metabolism suggests that acetylation may be a response to nutrient availability or the energy status of the cell. However, this and other hypotheses related to acetylation remain untested.

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“…Lysine acetylation has been detected following production in E. coli of recombinant therapeutic proteins, including the chemokine RANTES (d'Alayer et al, 2007), human basic fibroblast growth factor mutein (Suenaga et al, 1996), human carbonic anhydrase (Mahon et al, 2015), insulin lispro (Szewczak et al, 2015), interleukin-10 (Pflumm et al, 1997), interleukin-2 (Moya et al, 2002), interferon alpha (Takao et al, 1987), neurotropin-3 (Ross et al, 1996), and somatotropin (Violand et al, 1994). Nearly 30% of currently approved recombinant therapeutic proteins are produced in E. coli (Huang et al, 2012).…”

Section: Consequences For Pharmaceutical Productionmentioning

confidence: 99%

Bacterial protein acetylation: new discoveries unanswered questions

Wolfe

2015

Curr Genet

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Nε-acetylation is emerging as an abundant post-translational modification of bacterial proteins. Two mechanisms have been identified: one is enzymatic, dependent on an acetyltransferase and acetyl coenzyme A; the other is non-enzymatic and depends on the reactivity of acetyl phosphate. Some, but not most, of those acetylations are reversed by deacetylases. This review will briefly describe the current status of the field and raise questions that need answering.

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Isolation and Characterization of Acetylated Derivative of Recombinant Insulin Lispro Produced in Escherichia coli

Cited by 7 publications

References 38 publications

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media

Post-translational Protein Acetylation: An Elegant Mechanism for Bacteria to Dynamically Regulate Metabolic Functions

Bacterial protein acetylation: new discoveries unanswered questions

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