Expression, purification, and characterization of a His6-tagged glycerokinase from Pichia farinosa for enzymatic cycling assays in mammalian cells

Janke, R.; Genzel, Yvonne; Freund, Susann; Wolff, Michael W.; Grammel, Hartmut; Rühmkorf, Christine; Seidemann, J.; Wahl, A.; Reichl, Udo

doi:10.1016/j.jbiotec.2010.09.963

Cited by 2 publications

(2 citation statements)

References 43 publications

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“…Enzymes for analysis were purchased from Roche, except glycerol-3-phosphate oxidase from Streptococcus thermophilus, aspartate transaminase from pig heart, citrate synthase from porcine heart, diaphorase, glutamate dehydrogenase from bovine liver, creatine kinase, phosphoglycerate kinase, and glycerokinase from Escherichia coli (Sigma) and Pichia farinosa (Janke et al, 2010a).…”

Section: Reagents and Enzymesmentioning

confidence: 99%

Metabolic adaptation of MDCK cells to different growth conditions: Effects on catalytic activities of central metabolic enzymes

Janke¹,

Genzel²,

Händel³

et al. 2011

Biotech & Bioengineering

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Lactate and ammonia are the most important waste products of central carbon metabolism in mammalian cell cultures. In particular during batch and fed-batch cultivations these toxic by-products are excreted into the medium in large amounts, and not only affect cell viability and productivity but often also prevent growth to high cell densities. The most promising approach to overcome such a metabolic imbalance is the replacement of one or several components in the culture medium. It has been previously shown that pyruvate can be substituted for glutamine in cultures of adherent Madin-Darby canine kidney (MDCK) cells. As a consequence, the cells not only released no ammonia but glucose consumption and lactate production were also reduced significantly. In this work, the impact of media changes on glucose and glutamine metabolism was further elucidated by using a high-throughput platform for enzyme activity measurements of mammalian cells. Adherent MDCK cells were grown to stationary and exponential phase in six-well plates in serum-containing GMEM supplemented with glutamine or pyruvate. A total number of 28 key metabolic enzyme activities of cell extracts were analyzed. The overall activity of the pentose phosphate pathway was up-regulated during exponential cell growth in pyruvate-containing medium suggesting that more glucose-6-phosphate was channeled into the oxidative branch. Furthermore, the anaplerotic enzymes pyruvate carboxylase and pyruvate dehydrogenase showed higher cell specific activities with pyruvate. An increase in cell specific activity was also found for NAD(+)-dependent isocitrate dehydrogenase, glutamate dehydrogenase, and glutamine synthetase in MDCK cells grown with pyruvate. It can be assumed that the increase in enzyme activities was required to compensate for the energy demand and to replenish the glutamine pool. On the other hand, the activities of glutaminolytic enzymes (e.g., alanine and aspartate transaminase) were decreased in cells grown with pyruvate, which seems to be related to a decreased glutamine metabolism.

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Section: Reagents and Enzymesmentioning

confidence: 99%

Metabolic adaptation of MDCK cells to different growth conditions: Effects on catalytic activities of central metabolic enzymes

Janke¹,

Genzel²,

Händel³

et al. 2011

Biotech & Bioengineering

Self Cite

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“…Enzyme activities were quantified with three technical replicates each as described by Janke et al (Janke et al 2010a;Janke et al 2010b). In short, cells were harvested at a cell concentration of 1.3 × 10 6 cells/mL.…”

Section: Enzyme Activity Assaysmentioning

confidence: 99%

Tracking changes in adaptation to suspension growth for MDCK cells: cell growth correlates with levels of metabolites, enzymes and proteins

Pech

Rehberg

Janke

et al. 2021

Appl Microbiol Biotechnol

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Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth. Key points • Less and more efficient glucose utilization for suspension cell growth • Concerted alteration of metabolic enzyme activity and protein expression • Protein candidates to interfere glycolytic activity in MDCK cells

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Expression, purification, and characterization of a His6-tagged glycerokinase from Pichia farinosa for enzymatic cycling assays in mammalian cells

Cited by 2 publications

References 43 publications

Metabolic adaptation of MDCK cells to different growth conditions: Effects on catalytic activities of central metabolic enzymes

Metabolic adaptation of MDCK cells to different growth conditions: Effects on catalytic activities of central metabolic enzymes

Tracking changes in adaptation to suspension growth for MDCK cells: cell growth correlates with levels of metabolites, enzymes and proteins

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