Compositions, systems, and methods for high level expression of recombinant protein
AssigneeDuke University
InventorsMichael Lynch, Romel Menacho Melgar, Zhixia Ye, Eirik A. Moreb
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Abstract
Improved production of recombinant proteins in E. coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in stationary phase. The process also provides an optimized autoinduction media, enabling routine batch production at various culture volumes where cells densities routinely reach ˜5-7 g cell dry weight per liter and offer protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation.
Claims15
A method of expression of a protein comprising:
- providing an E. coli genetically modified microorganism that is obtained by transforming a microorganism strain with a plasmid, the plasmid comprising a phoB regulated promoter operatively linked to a nucleic acid encoding the expressed protein;
- and growing the genetically modified microorganism in a batch media to obtain a biomass and express the protein, wherein the batch media enables growth of the genetically modified microorganism in the presence of phosphate in the batch media and autoinduction of protein expression upon depletion of phosphate from the batch media, wherein the batch media contains greater than 5.0 g/L and less than 15.0 g/L of a combination of yeast extract and casamino acids, and the batch media contains from about 15 mM to about 400 mM (NH4)2SO4, ammonium sulfate, wherein the microorganism does not produce the byproduct acetic acid during growth, and
- wherein the expressed protein is between about 10 and about 55% of the total cellular protein content of the genetically modified microorganism.
Dependencies are inferred from the claim text.