Limitations for purification of murine interleukin-18 when expressed as a fusion protein containing the FLAG peptide.
As a strategy to purify recombinant murine Interleukin (IL)-18, we cloned the mature coding region of this protein into the pFLAG-1 expression system. The intent was to use the FLAG peptide "tag" as an amino terminal addition to IL-18 so that purification of this fusion protein (FLAG-IL-18) on anti-FLAG antibody affinity columns could be performed. While significant amounts of recombinant IL-18 were present in E. coli lysates, only a small portion of this material could be recovered on immunoaffinity columns conjugated with an anti-FLAG antibody. Surprisingly, the majority of recombinant IL-18 present in E. coli (strain JM83) bacterial lysates did not contain the FLAG peptide and therefore did not bind to immunoaffinity columns conjugated with an anti-FLAG antibody. However, we found that the BL21 strain of E. coli, which has reduced endogenous protease activity, could express the majority of recombinant IL-18 as the fusion protein, FLAG-IL-18. Taken together, these studies show that it is necessary to consider whether protease sites formed at the FLAG-protein junction can be easily cleaved by the bacterial strain used to express the fusion protein.
University of North Carolina at Charlotte, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006868:Hydrolysis; D020382:Interleukin-18 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D020382:Interleukin-18; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide; D010450:Endopeptidases