Expression fusion protein

Fusion protein pull-down assays involve the overexpression of bait and/or fusion proteins in bacteria. Often, the expressed fusion proteins are localized in occlusion bodies and not readily soluble under nondenaturing conditions. The expressed proteins can be extracted using urea, sonication, sodium dodecyl sulfate (SDS), or a combination of all the three. The net result is the denaturation of the recombinant protein and it may need to be refolded if the interaction domain is conformationally dependent. A major advantage of the pull-down assay is that high concentrations of proteins can be easily generated thus favoring protein association for a reversible equilibrium between two proteins. 

The methods described are suitable for use with immunogens derived from a variety of sources, for example, bacterially expressed fusion proteins, baculovirus-expressed proteins or synthetic peptides conjugated to suitable carrier proteins (see Chapter 2). Antibody responses to peptide immunogens often differ from those where the immunogen is a larger macromolecule in that maximal antipeptide titres (which arise rapidly after two to three immunizations) do not always coincide with maximal titres against the intact... 

Chen et al. (2008) have expressed a codon-optimized form of the CM4 peptide in E. coli. In this case, two alternative expression/purification systems were examined namely the widely used glutathione-S-transferase (GST) and a chitin-binding domain (CBD) system with associated intein splicing (New England Biolabs Inc.). The GST system failed to allow recovery of expressed fusion protein. In contrast, the CBD/intein system allowed the recovery of 110 mg/L fusion protein with a final RP-HPLC purification step yielding 2.1 mg/L of pure peptide which displayed antimicrobial activity against E. coli Ki2D3i and Salmonella. 

The import of proteins into in v/fra-assembled nuclei has been followed with reporter proteins, such as a fluorescently labeled BSA conjugated with the nuclear localization sequence (NLS) of SV40 T antigen. We have produced a bacterially expressed fusion protein that contains the sequence for green fluorescent protein (GFP) (Chalfie et al., 1994) in frame with the SV40 T antigen NLS (X. C. Wu... 

Scheme 7.34 Small-molecule-based optical highlighter 39 for expressing fusion protein. (Reproduced by permission of American Chemical Society.

Fig. I. Gel-shift analysis of a retinoid responsive element of the P-RAR promoter with DR5 configuration that binds RAR and RXR synergistically. The probes were endlabeled with Klenow enzyme and incubated with the bacterially expressed fusion proteins fluRARa [containing the hemaglutinin epitope (14)] and mycRXRa [containing the c-myc 9E10 epitope (12). Coincubation with anti-flu and anti-niyc antibodies respectively results in a disappearance of the heterodimeric complex and a supershift. In contrast, 15C5—a nonrelated monoclonal antibody—does not affect the complex.

Prepare two chitin column for affinity-chromatographic purification of expressed fusion protein. Therefore, fill slurry of chitin beads in a column and allow them to decant (final bed volume 15 mL). Wash chitin beads with water (20 bed volumes) and equilibrate with column buffer containing 3 M urea (10 bed volumes). 

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