Antibiotic-free selection in E. coli: new considerations for optimal design and improved production.
BACKGROUND
The increasing regulatory requirements to which biological agents are subjected will have a great impact in the field of industrial protein expression and production. There is an expectation that in a near future, there may be "zero tolerance" towards antibiotic-based selection and production systems. Besides the antibiotic itself, the antibiotic resistance gene is an important consideration. The complete absence of antibiotic-resistance gene being the only way to ensure that there is no propagation in the environment or transfer of resistance to pathogenic strains.


RESULTS
In a first step, we have designed a series of vectors, containing a stabilization element allowing a complete elimination of antibiotics during fermentation. Vectors were further improved in order to include alternative selection means such as the well known poison/antidote stabilization system. Eventually we propose an elegant positive pressure of selection ensuring the elimination of the antibiotic-resistance gene through homologous recombination. In addition, we have shown that the presence of an antibiotic resistance gene can indirectly reduce the amount of expressed protein, since even in absence of selection pressure the gene would be transcribed and account for an additional stress for the host during the fermentation process.


CONCLUSIONS
We propose a general strategy combining plasmid stabilization and antibiotic-free selection. The proposed host/vector system, completely devoid of antibiotic resistance gene at the end of construction, has the additional advantage of improving recombinant protein expression and/or plasmid recovery.
Peubez
Isabelle
I
Research Department, Sanofi Pasteur, Marcel Mérieux, 69280 Marcy l'Etoile, France.
Chaudet
Nicolas
N
Mignon
Charlotte
C
Hild
Géraldine
G
Husson
Stéphanie
S
Courtois
Virginie
V
De Luca
Karelle
K
Speck
Denis
D
Sodoyer
Régis
R
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D005822:Genetic Vectors; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068355:CcdA protein, Bacteria; C068356:CcdB protein, Plasmid F; D011994:Recombinant Proteins
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