Fusion protein techniques

ELP fusion protein (Fig. 9, center). It was shown that picomolar levels of ELP fusion proteins could be purified via ELP coaggregation [47, 48]. The value of this technique was shown by the purification of low levels of ELP fused to an anti-atrazine antibody [49]. 

In principle, the use of addressable, pooled GST fusion proteins could be used to identify proteins associated with any biochemical activity, assuming that the fusion protein is soluble, folded and functional. The method has the additional advantage that, once the GST fusion clones are constructed, it is a rapid technique. The authors state that only two weeks are required to purify the 64 pools and the assays can be accomplished in a day (Martzen et al., 1999). In addition, the method is sensitive because only 96 recombinant proteins are assayed at one time in contrast to the use of cell lysates where thousands of proteins are present. This leads to a much higher concentration of each protein, which greatly facilitates detection of a biochemical activity (Martzen et al., 1999). 

Carcinoembryonic antigen (CEA) 2001 A Circulating CEA in human sera 92.3% of patients with high CEA levels and 50% of patients with normal CEA levels were found positive Ren et al. [18], use of a bispecific CEAScFv-streptavidin fusion protein-based immuno-PCR technique (see Section 2.1.1)... 

Ren J, Ge L, Li Y, Bai J, Liu WC, Si XM. Detection of circulating CEA molecules in human sera and leukopheresis of peripheral blood stem cells with E. coli expressed bispecific CEAScFv-streptavidin fusion protein-based immuno-PCR technique. Ann NY Acad Sci 2001 945 116-118. 

Lindner P, Bauer K, Krebber A, Nieba L, Kremmer E, Krebber C, Honegger A, Klinger B, Mocikat R, Pluckthun A, Specific detection of his-tagged proteins with recombinant anti-His tag scFv-phosphatase or scFv-phage fusions, Bio techniques, 22 140-149, 1997. 

Future applications of miniaturized expanded-bed columns could be the detection of fusion proteins from crude fermentation broth, which gives an alternative to the conventional analytical techniques which require a series of sample cleanup procedure prior to analysis. 

You have isolated a protein of interest tagged with the GST domain. At this point, the fusion protein is bound to the glutathione Sepharose 4B resin and the unbound proteins have been washed away. Describe, in detail, two different techniques that you could use to isolate the protein without the affinity tag using only thrombin, glutathione Sepharose 4B, and reduced glutathione. 

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