Expression and purification of lacZ and trpE fusion proteins.
Fusion proteins are commonly used as a source of antigen for producing antibodies and in many cases can be useful for biochemical analyses. This unit describes two widely used expression systems for producing large amounts of proteins in E. coli. One system expresses lacZ fusions using the pUR series of vectors and the other expresses trpE fusions using the pATH vectors. The gene of interest is first subcloned into either a pUR or pATH vector in the correct reading frame. The correct transformant is selected, grown, and then induced with either IPTG or IAA. Sonication of cells in the presence of protease inhibitors is used to prepare extracts containing both types of fusion proteins, as well as other types of proteins overexpressed in E. coli. The extracts are checked for the presence of fusion protein on an SDS-polyacrylamide gel.
Hoey
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University of California, Berkeley, California, USA.
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase