Fractional factorial approach combining 4 Escherichia coli strains, 3 culture media, 3 expression temperatures and 5 N-terminal fusion tags for screening the soluble expression of recombinant proteins.
Producing recombinant proteins in Escherichia coli (E. coli) is generally performed using a trial and error approach with the different expression variables being tested independently from each other. As a consequence, variable interactions are lost which makes the trial and error approach quite time-consuming. In this paper, we report how switching from a trial and error to a fractional factorial approach allows testing in less than 2 weeks four expression variables (E. coli strains, culture media, expression temperatures and N-terminal fusion tags) in a single experiment. The method, called "Fusion-InFFact", was validated using four test proteins. In all cases, Fusion-InFFact allowed finding conditions for expressing high yields of soluble proteins. The method was originally set-up for high throughput structural genomics programs, but can be used in any recombinant protein expression project.
Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 7257 CNRS and Aix-Marseille University, Marseille, France.
Christophe
C
Noguère
Anna M
AM
Larsson
Jean-Christophe
JC
Guyot
Christophe
C
Bignon
D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003470:Culture Media / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D023281:Genomics; D006639:Histidine / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013696:Temperature
D003470:Culture Media; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine