- Using secretion to solve a solubility problem: high-yield expression in Escherichia coli and purification of the bacterial glycoamidase PNGase F.
Using secretion to solve a solubility problem: high-yield expression in Escherichia coli and purification of the bacterial glycoamidase PNGase F.
Protein expression and purification (2002-01-29)
Trevor Loo, Mark L Patchett, Gillian E Norris, J Shaun Lott
PMID11812228
ABSTRAKT
PNGase F is a widely used deglycosidase, secreted in small amounts by the gram-negative bacterium Flavobacterium meningosepticum. We have designed a T7 promoter-based Escherichia coli expression system to provide a high-yield source of recombinant enzyme. When expressed intracellularly, the enzyme was produced in a largely insoluble state. However, when expressed as a fusion with the leader sequence from the ompA gene, hexahistidine-tagged PNGase F was efficiently processed and exported to the E. coli periplasm. Single-step purification using immobilized metal affinity chromatography yielded 8 mg of pure enzyme per liter of culture, which is fully active on a range of protein and peptide substrates.