Enhancing the selective extracellular location of a recombinant E. coli domain antibody by management of fermentation conditions
| Autor: | Mike Hoare, Ioannis Voulgaris, Gary Finka, Mark Uden |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Lysis
Gram-negative bacteria medicine.disease_cause Applied Microbiology and Biotechnology Antibodies Permeability Periplasmic release medicine Extracellular Escherichia coli Polyethyleneimine Domain antibody biology E. coli General Medicine biology.organism_classification Biotechnological Products and Process Engineering Recombinant Proteins Culture Media Protein Transport Biochemistry Fermentation Nucleic acid Bacterial outer membrane Homogenization (biology) Biotechnology |
| Zdroj: | Applied Microbiology and Biotechnology |
| ISSN: | 1432-0614 0175-7598 |
| Popis: | The preparation of a recombinant protein using Escherichia coli often involves a challenging primary recovery sequence. This is due to the inability to secrete the protein to the extracellular space without a significant degree of cell lysis. This results in the release of nucleic acids, leading to a high viscosity, difficulty to clarify, broth and also to contamination with cell materials such as lipopolysaccharides and host cell proteins. In this paper, we present different fermentation strategies to facilitate the recovery of a V H domain antibody (13.1 kDa) by directing it selectively to the extracellular space and changing the balance between domain antibody to nucleic acid release. The manipulation of the cell growth rate in order to increase the outer cell membrane permeability gave a small ~1.5-fold improvement in released domain antibody to nucleic acid ratio without overall loss of yield. The introduction during fermentation of release agents such as EDTA gave no improvement in the ratio of released domain antibody to nucleic acid and a loss of overall productivity. The use of polyethyleneimine (PEI) during fermentation was with the aim to (a) permeabilise the outer bacterial membrane to release selectively domain antibody and (b) remove selectively by precipitation nucleic acids released during cell lysis. This strategy resulted in up to ~4-fold increase in the ratio of domain antibody to soluble nucleic acid with no reduction in domain antibody overall titre. In addition, a reduction in host cell protein contamination was achieved and there was no increase in endotoxin levels. Similar results were demonstrated with a range of other antibody products prepared in E. coli. |
| Databáze: | OpenAIRE |
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