Using secretion to solve a solubility problem: high-yield expression in Escherichia coli and purification of the bacterial glycoamidase PNGase F.
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.
Loo
Trevor
T
Institute of Molecular Biosciences, Massey University, Private Bag 11-222, Palmerston North, New Zealand.
Patchett
Mark L
ML
Norris
Gillian E
GE
Lott
J Shaun
JS
D000581:Amidohydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006031:Glycosylation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
D001425:Bacterial Outer Membrane Proteins; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; C078167:OMPA outer membrane proteins; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase