Continuous Immobilized Recombinant Protein Production from E. coli Capable of Selective Protein Excretion: A Feasibility Study.
Currently genetically engineered bacteria are economically attractive only for the production of high-value, low-volume-proteins. For this technology to be useful for the manufacture of high volume proteins new bioprocessing concepts are necessary. A potential approach is to construct a plasmid containing an inducible promoter and a hybrid gene containing a signal sequence fused to the gene for the protein to be produced. The plasmid is transformed into Escherichia coli. The plasmid-host interaction is such that the host becomes "leaky" upon introduction. Since the signal sequence allows the recombinant protein to pass the cytoplasmic membrane, a "leaky cell" can allow selective excretion of the desired protein. If the "leaky" cell is immobilized and maintained in a resting state, the sustained continuous production of the target protein is possible. To test this concept we have used plasmid pKK in E. coli RB791 with beta-lactamase as the target protein. Production levels of at least 390 units beta-lactamase/L-h can be sustained for at least 120 days. Peak productivities of 10,000 unitslL-h have been obtained. The beta-lactamase constitutes approximately 40% of the protein in solution. Such systems could reduce recombinant protein production costs due both to the continuous nature of the bioreactor and the improved ease of protein recovery.
Georgiou
G
G
School of Chemical Engineering University Ithaca, New York 14853.
Chalmers
J J
JJ
Shuler
M L
ML
Wilson
D B
DB