High-level expression and efficient recovery of ubiquitin fusion proteins from Escherichia coli.
The transition in growth and induction of bacterial cultures expressing recombinant proteins from the laboratory bench to the pilot scale for production has been performed successfully for several ubiquitin fusion-expressing clones. Increased protein turnover and decreased metabolic efficiency of Escherichia coli at high cell densities are often responsible for failures in fermenter cultures. Current data indicate that (1) yields in shaker flask cultures are directly scalable to a 10 L fermenter, (2) higher cell densities actually augment the specific yield of ubiquitin fusion proteins, (3) an in vivo heat shock during fermentation increases the ubiquitin fusion yield, which provides an initial separation step in the fermenter, and (4) the ubiquitin fusion expression clone makes at least 3-fold more total protein, including host proteins, than the parent strain of E. coli. A series of three fermentations was performed, using the model strain, with varied temperature shift protocols. These fermentations showed that a maximal heat shock of 12 degrees C (from 30 to 42 degrees C), initiated simultaneously with induction, gave a maximal specific yield (over 90% of the total soluble protein by densitometry, 709 mg/L by protein assay), in which the recoverable ubiquitin fusion product comprised 16% of the wet weight of the cell paste. These results illustrate the enormous potential of ubiquitin fusion technology for the economical production of peptides, even in a 10 L fermenter.
Pilon
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AL
Proteinix Company, Gaithersburg, Maryland 20877, USA.
Yost
P
P
Chase
T E
TE
Lohnas
G L
GL
Bentley
W E
WE
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D018869:Heat-Shock Response; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature; D014452:Ubiquitins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins