Expression of Fusion Proteins

Fusion protein Origin MW [kD] Ligand for affinity purification 

Intein/chitin domain Saccharomyces cerevisiae/ Bacillus circulans 55 chitin 

APTG p-aminophenyl-/3-D-thiogalactoside IgG immunoglobulin G. Source Lottspeich, 1998. 

The separation of the target protein from the fusion protein can be performed chemically or enzymatically. The basis for chemical cleavage is acid or base stability of the target protein. Enzymatic separation by proteases is highly specific but its efficiency can be decreased by limited access to the part of the amino acid sequence required for proteolysis. 

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This method combines the use of antibodies and the expression of fusion proteins to deliver an approach which can be applied in a very generic way. The protein of interest is expressed in a cell-line with an epitope-tag which is recognised by an antibody (Figure 1C). Many different cDNA s can be fused to the same tag allowing purification of many different complexes in parallel using the same strategy. Many... 

In summary, although the application of Y3H may be limited in some scenarios, most of these are likely to be rare events. The most limiting factor is likely the requirement for expression of fusion proteins that are able to translocate into the nucleus of yeast cells while retaining a properly folded small molecule binding domain. This may, however, not be an issue with many proteins, because of their modular structure. A modular structure favors proper folding of a binding domain, even when it is expressed in isolation or as part of a hybrid fusion protein. Thus, the use of complex cDNA libraries, which contain multiple fusion variants of a particular protein, is preferable and will decrease the occurrence of false negatives. 

Reagents and equipment for PCR, restriction digests, and DNA Hgations. Reagents to vahdate expression of fusion proteins, such as antibodies for flow cytometry and/or Western blotting. 

Hoffinann I, Jemeren F, Oliw EH (2013) Expression of fusion proteins of Aspergillus terreus reveals a novel allene oxide synthase. J Biol Chem 288 11459 11469... 

The fl-galactosidase complementation assay has also been adapted for use in mammalian cells (Rossi et al., 1997). The availability of fluorescent substrates for (3-galactosidase allows for fluorescence microscopy and FACS analysis of mammalian cells expressing the fusion proteins of interest. Therefore, similar to the mDHFR system, fl-galactosidase complementation assays may prove useful for genome-scale studies of protein-protein interactions in mammalian cells. 

Fig. 8.11 (A and B) Expression and purification of insulin-polymer fusion protein detected in copper (A) and Coomassie (B) stained gels. The same gel was first stained with copper, destained and restained with Coomassie R-250. Lane 1 Prestained marker Lane 2 Purified extract of polymer-insulin fusion protein from the chloroplast vector pSBL-OC-40Pris Lane 3 Reverse orientation of fusion protein from pSBL-OC-40Pris Lane 4 Purified extract of polymer-insulin fusion protein from the chloroplast vector pLD-OC-40Pris ...

As an alternative to targeting brain tumours which express the TfR, the transferrin approach can be used for the delivery of fusion proteins which bind to pharmacological receptors inside the central nervous system. An example of this is the construct consisting of nerve growth factor (NGF) and transferrin described in Section 11.8.2.3. The transferrin moiety in this type of construct will enable it to enter the brain, upon which the drug moiety will act by binding to its receptor. This approach seems especially suitable for compounds that cannot pass the blood-brain barrier, such as peptides and other hydrophilic substances. 

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