Large-scale production of citrate synthase from a cloned gene

1982 ◽  
Vol 60 (12) ◽  
pp. 1143-1147 ◽  
Author(s):  
Harry W. Duckworth ◽  
Alexander W. Bell
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Citrate Synthase ◽  
Specific Activity ◽  
Tetracycline Resistance ◽  
Scale Production ◽  
Ampicillin Resistance ◽  
Colicin E1 ◽  
Large Scale Production ◽  
Cloned Gene

Starting with a colicin E1 resistance recombinant plasmid which contains gltA, the gene for citrate synthase in Escherichia coli, we have constructed an ampicillin-resistance plasmid containing the gltA region as a 2.9-kilobase-pair insert in the tetracycline-resistance region of pBR322. Escherichia coli HB101 harbouring this plasmid, when grown on rich medium containing ampicillin, contains citrate synthase as about 8% of its soluble protein. The enzyme has been purified from this rich source and is identical to the chromosomal enzyme prepared previously in every property tested, except for specific activity, which is 64 U∙mg−1 as compared with 45–50 U∙mg−1 previously obtained. The N-terminal sequences of both enzymes are reported, and they are identical up to residue 16 at least. The overall yield of pure enzyme, starting with the cells grown in 15 L of medium, is 600–800 mg.

scholarly journals Efficient Production of l-Ribose with a Recombinant Escherichia coli Biocatalyst

2008 ◽  
Vol 74 (10) ◽  
pp. 2967-2975 ◽  
Author(s):  
Ryan D. Woodyer ◽  
Nathan J. Wymer ◽  
F. Michael Racine ◽  
Shama N. Khan ◽  
Badal C. Saha
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Active Form ◽  
Apium Graveolens ◽  
Efficient Production ◽  
Scale Production ◽  
Gene Encoding ◽  
Large Scale Production ◽  
One Step ◽  
Rare Sugars

ABSTRACT A new synthetic platform with potential for the production of several rare sugars, with l-ribose as the model target, is described. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically constructed for optimal expression in Escherichia coli. This MDH enzyme catalyzes the interconversion of several polyols and their l-sugar counterparts, including the conversion of ribitol to l-ribose. Expression of recombinant MDH in the active form was successfully achieved, and one-step purification was demonstrated. Using the created recombinant E. coli strain as a whole-cell catalyst, the synthetic utility was demonstrated for production of l-ribose, and the system was improved using shaken flask experiments. It was determined that addition of 50 to 500 μM ZnCl2 and addition of 5 g/liter glycerol both improved production. The final levels of conversion achieved were >70% at a concentration of 40 g/liter and >50% at a concentration of 100 g/liter. The best conditions determined were then scaled up to a 1-liter fermentation that resulted in 55% conversion of 100 g/liter ribitol in 72 h, for a volumetric productivity of 17.4 g liter−1 day−1. This system represents a significantly improved method for the large-scale production of l-ribose.

Development of a Large Scale Production Process for BAX 855, a PEGylated rFVIII Product with Prolonged Half-Life

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4362-4362
Author(s):  
Jürgen Siekmann ◽  
Purtscher Martin ◽  
Oliver Zöchling ◽  
Robert Kellerer ◽  
Hanspeter Rottensteiner ◽  
...  
Keyword(s):  
Manufacturing Process ◽  
Large Scale ◽  
Specific Activity ◽  
Drug Product ◽  
Scale Production ◽  
Drug Products ◽  
Large Scale Production ◽  
Bulk Drug ◽  
Recombinant Fviii ◽  
Conjugation Process

Abstract Abstract 4362 Baxter and Nektar have developed BAX 855, a PEGylated form of Baxter’s recombinant FVIII (rFVIII) product based on the ADVATE™ manufacturing process. The conjugation process for preparing BAX 855 uses proprietary stable PEGylation from Nektar Therapeutics. Similar Nektar technology has been successfully employed for marked and licensed PEGylated drug products and drugs in clinical use. The manufacturing process for BAX 855 comprises several steps, including chromatographic purification on a MacroCap SP resin, a strong cation exchanger, which allows the fractionation of species with different PEGylation degrees and concentration of the conjugate collected by an ultra- / diafiltration step leading to the pre-formulated bulk drug substance (BDS). Final formulation of the BDS includes a filling and lyophilization step to obtain the final drug product. The process described is suited to manufacture BAX 855 in gram scale and showed a good batch to batch consistency, ensuring an equivalent product quality for each batch. BAX 855 manufactured by this process has a specific activity similar to that of rFVIII in ADVATE™ and PEGylation degrees in the narrow range of 2 to 3 mols PEG / mol rFVIII. SDS-PAGE and Western blot analysis of BAX 855 confirm the stable PEGylation and demonstrate an increase in the molecular weight of the various FVIII domains. PK studies in different species display longer survival of BAX 855 compared to ADVATE™. Disclosures: Siekmann: Baxter Innovations GmbH: Employment. Martin:Baxter Innovations GmbH: Employment. Zöchling:Baxter Innovations GmbH: Employment. Kellerer:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Mitterer:Baxter Innovations GmbH: Employment. Bossard:Nektar Therapeutics: Employment. Phillips:Nektar Therapeutics: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.

scholarly journals Anti-L. donovani Activity in Macrophage/Amastigote Model of Palmarumycin CP18 and its Large Scale Production

2014 ◽  
Vol 9 (1) ◽  
pp. 1934578X1400900
Author(s):  
Humberto E. Ortega ◽  
Eliane de Morais Teixeira ◽  
Ana Rabello ◽  
Sarah Higginbotham ◽  
Luis Cubilla-Ríos
Keyword(s):  
Large Scale ◽  
Leishmania Donovani ◽  
Specific Activity ◽  
Fermentation Broth ◽  
Fungal Culture ◽  
Scale Production ◽  
Corn Meal ◽  
H Nmr ◽  
Solid Media ◽  
Large Scale Production

Palmarumycin CP18, isolated from an extract of the fermentation broth and mycelium of the Panamanian endophytic fungus Edenia sp., was previously reported with strong and specific activity against Leishmania donovani. Here we report that when the same strain was cultured on different solid media – Harrold Agar, Leonian Agar, Potato dextrose Agar (PDA), Corn Meal Agar, Honey Peptone Agar, and eight vegetables (V8) Agar – in order to determine the optimal conditions for isolation of palmarumycin CP18, no signal for this compound was observed in any of the 1H NMR spectra of fractions obtained from these extracts. However, one extract, prepared from the fungal culture in PDA contained significant amounts of CJ-12,372, a possible biosynthetic precursor of palmarumycin CP18. Edenia sp. was cultivated on a large scale on PDA and CJ-12,372 was converted to palmarumycin CP18 by oxidation of its p-hydroquinone moiety with DDQ in dioxane. Palmarumycin CP18 showed anti-leishmanial activity against L. donovani in a macrophage/amastigote model, with IC50 values of 23.5 μM.

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