scholarly journals Conversion of spent CHO cell culture media waste to new fermentation feed efficiently supports production of recombinant protein by Escherichia coli

2021 ◽  
Author(s):  
Ciara Lynch ◽  
David J O'Connell
Keyword(s):  
Cell Culture ◽  
Recombinant Protein ◽  
Culture Media ◽  
Mass Spectrometry Analysis ◽  
Microbial Fermentation ◽  
Differential Protein Expression ◽  
Cell Culture Media ◽  
Spectrometry Analysis ◽  
E Coli ◽  
Rich Media

Deriving new value from waste streams is a central aim of the circular bioeconomy. In this study we investigate whether chemically defined spent media (CDSM) waste from cell culture bioprocess can be effectively recycled and used as a feed in microbial fermentation to produce new recombinant protein products. Our results show that 1) CDSM supplemented with 2% glycerol supported a specific growth rate of E. coli cultures equivalent to that achieved using a nutritionally rich media (LB) used as a baseline reference. 2) The amount of recombinant protein produced following induction in an expression screen was approximately two-fold higher in the CDSM fed cultures than that of baseline. 3) Mass spectrometry analysis of the proteome of E. coli cultures fed in CDSM revealed a greater or lesser differential protein expression pattern depending on supplementation conditions. Further, in a 16 hr fermentation the optimised CDSM-fed culture delivered a protein yield of more than double that achieved by the baseline media. We conclude that spent cell culture media, which represents millions of litres of waste generated by the bioprocessing industry annually, has the potential to be a valuable feed resource for the production of recombinant proteins in secondary microbial fermentation.

scholarly journals Evidence for Direct Protein-Protein Interaction between Members of the Enterobacterial Hha/YmoA and H-NS Families of Proteins

2002 ◽  
Vol 184 (3) ◽  
pp. 629-635 ◽  
Author(s):  
J. M. Nieto ◽  
C. Madrid ◽  
E. Miquelay ◽  
J. L. Parra ◽  
S. Rodríguez ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Protein A ◽  
Nitrilotriacetic Acid ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
E Coli ◽  
Protein Protein Interaction ◽  
New Family ◽  
Missense Mutant ◽  
First Time

ABSTRACT Escherichia coli nucleoid-associated H-NS protein interacts with the Hha protein, a member of a new family of global modulators that also includes the YmoA protein from Yersinia enterocolitica. This interaction has been found to be involved in the regulation of the expression of the toxin α-hemolysin. In this study, we further characterize the interaction between H-NS and Hha. We show that the presence of DNA in preparations of copurified His-Hha and H-NS is not directly implicated in the interaction between the proteins. The precise molecular mass of the H-NS protein retained by Hha, obtained by mass spectrometry analysis, does not show any posttranslational modification other than removal of the N-terminal Met residue. We constructed an H-NS-His recombinant protein and found that, as expected, it interacts with Hha. We used a Ni2+-nitrilotriacetic acid agarose method for affinity chromatography copurification of proteins to identify the H-NS protein of Y. enterocolitica. We constructed a six-His-YmoA recombinant protein derived from YmoA, the homologue of Hha in Y. enterocolitica, and found that it interacts with Y. enterocolitica H-NS. We also cloned and sequenced the hns gene of this microorganism. In the course of these experiments we found that His-YmoA can also retain H-NS from E. coli. We also found that the hns gene of Y. enterocolitica can complement an hns mutation of E. coli. Finally, we describe for the first time systematic characterization of missense mutant alleles of hha and truncated Hha′ proteins, and we report a striking and previously unnoticed similarity of the Hha family of proteins to the oligomerization domain of the H-NS proteins.

Cell culture media: NMR approaches to evaluating quality and nutrient consumption

Planta Medica ◽  
2016 ◽  
Vol 81 (S 01) ◽  
pp. S1-S381
Author(s):  
KB Killday ◽  
AS Freund ◽  
C Fischer ◽  
KL Colson
Keyword(s):  
Cell Culture ◽  
Culture Media ◽  
Cell Culture Media ◽  
Nutrient Consumption

The Influence of Glycosylation on the Catalytic and Fibrinolytic Properties of Pro-Urokinase

1992 ◽  
Vol 68 (05) ◽  
pp. 539-544 ◽  
Author(s):  
Catherine Lenich ◽  
Ralph Pannell ◽  
Jack Henkin ◽  
Victor Gurewich
Keyword(s):  
Escherichia Coli ◽  
Mammalian Cell ◽  
Human Gene ◽  
Culture Media ◽  
Mammalian Cell Culture ◽  
Glycosylation Site ◽  
Clot Lysis ◽  
Cell Culture Media ◽  
E Coli ◽  
The Uk

SummaryWe previously found that human pro-UK expressed in Escherichia coli is more active in fibrinolysis than recombinant human pro-UK obtained from mammalian cell culture media. To determine whether this difference is related to the lack of glycosylation of the E. coli product, we compared the activity of E. coli-derived pro-UK [(-)pro-UK] with that of a glycosylated pro-UK [(+)pro-UK] and of a mutant of pro-UK missing the glycosylation site at Asn-302 [(-) (302) pro-UK]. The latter two pro-UKs were obtained by expression of the human gene in a mammalian cell. The nonglycosylated pro-UKs were activated by plasmin more efficiently (≈2-fold) and were more active in clot lysis (1.5-fold) than the (+)pro-UK. Similarly, the nonglycosylated two-chain derivatives (UKs) were more active against plasminogen and were more rapidly inactivated by plasma inhibitors than the (+)UK.These findings indicate that glycosylation at Asn-302 influences the activity of pro-UK/UK and could be the major factor responsible for the enhanced activity of E. coli-derived pro-UK.

Selection and preliminary application of DNA aptamer targeting A549 excreta in cell culture media

2021 ◽  
pp. 106811
Author(s):  
Yuanbin Guo ◽  
Ming Shi ◽  
Xiujuan Liu ◽  
Huagang Liang ◽  
Liming Gao ◽  
...  
Keyword(s):  
Cell Culture ◽  
Culture Media ◽  
Dna Aptamer ◽  
Cell Culture Media ◽  
Preliminary Application

scholarly journals Benchmarking of commercially available CHO cell culture media for antibody production

2015 ◽  
Vol 99 (11) ◽  
pp. 4645-4657 ◽  
Author(s):  
David Reinhart ◽  
Lukas Damjanovic ◽  
Christian Kaisermayer ◽  
Renate Kunert
Keyword(s):  
Cell Culture ◽  
Antibody Production ◽  
Culture Media ◽  
Cho Cell ◽  
Cell Culture Media ◽  
Cho Cell Culture

scholarly journals Intestinal and Hepatic Uptake of Dietary Peroxidized Lipids and Their Decomposition Products, and Their Subsequent Effects on Apolipoprotein A1 and Paraoxonase1

Antioxidants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1258
Author(s):  
Xueting Jiang ◽  
Pragney Deme ◽  
Rajat Gupta ◽  
Dmitry Litvinov ◽  
Kathryn Burge ◽  
...  
Keyword(s):  
Cell Culture ◽  
Culture Media ◽  
Degradation Products ◽  
Apolipoprotein A1 ◽  
Paraoxonase 1 ◽  
Pon1 Activity ◽  
Atherosclerotic Cardiovascular Disease ◽  
Metabolic Fate ◽  
Cell Culture Media ◽  
Decomposition Products

Both pro- and antiatherosclerotic effects have been ascribed to dietary peroxidized lipids. Confusion on the role of peroxidized lipids in atherosclerotic cardiovascular disease is punctuated by a lack of understanding regarding the metabolic fate and potential physiological effects of dietary peroxidized lipids and their decomposition products. This study sought to determine the metabolic fate and physiological ramifications of 13-hydroperoxyoctadecadienoic acid (13-HPODE) and 13-HODE (13-hydroxyoctadecadienoic acid) supplementation in intestinal and hepatic cell lines, as well as any effects resulting from 13-HPODE or 13-HODE degradation products. In the presence of Caco-2 cells, 13-HPODE was rapidly reduced to 13-HODE. Upon entering the cell, 13-HODE appears to undergo decomposition, followed by esterification. Moreover, 13-HPODE undergoes autodecomposition to produce aldehydes such as 9-oxononanoic acid (9-ONA). Results indicate that 9-ONA was oxidized to azelaic acid (AzA) rapidly in cell culture media, but AzA was poorly absorbed by intestinal cells and remained detectable in cell culture media for up to 18 h. An increased apolipoprotein A1 (ApoA1) secretion was observed in Caco-2 cells in the presence of 13-HPODE, 9-ONA, and AzA, whereas such induction was not observed in HepG2 cells. However, 13-HPODE treatments suppressed paraoxonase 1 (PON1) activity, suggesting the induction of ApoA1 secretion by 13-HPODE may not represent functional high-density lipoprotein (HDL) capable of reducing oxidative stress. Alternatively, AzA induced both ApoA1 secretion and PON1 activity while suppressing ApoB secretion in differentiated Caco-2 cells but not in HepG2. These results suggest oxidation of 9-ONA to AzA might be an important phenomenon, resulting in the accumulation of potentially beneficial dietary peroxidized lipid-derived aldehydes.

Characterization of the impact of classical cell‐culture media on the response of electrochemical sensors

2021 ◽  
Author(s):  
Ayman Chmayssem ◽  
Lauriane Petit ◽  
Nicolas Verplanck ◽  
Véronique Mourier ◽  
Séverine Vignoud ◽  
...  
Keyword(s):  
Cell Culture ◽  
Electrochemical Sensors ◽  
Culture Media ◽  
Cell Culture Media ◽  
The Impact
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