Fused proteins

Figure 6.2. Domain fusion method to detect functional linkages between proteins. If two proteins, A and B, from a certain species are expressed as a single fused protein within another species, the proteins A and B are proposed to be functionally linked. Because the fusion protein contains homology to both A and B, it is termed a Rosetta Stone sequence. Specific examples from domain fusion analyses are shown. Figure adapted from Eisenberg et al. (2000) and Marcotte et al. (1999).

Lastly, pharmacogenomics could provide new tools for the design of more specific and active CNS pharmaceuticals. The efficacy of a broad spectrum of neuro-pharmaceutical drugs is often complicated by their inability to reach their site of action because of the BBB. One way to overcome this is to use carrier-mediated transport at the luminal and/or abluminal membranes of the endothelial cells of the BBB. This will provide a physiologically based drug delivery strategy for the brain by designing new chemical entities or fused proteins that can cross the BBB via these transporters. 

Use of a fusion protein to obtain crystals suitable for X-ray analysis crystallization of a GST-fused protein containing the DNA-binding domain of DNA replication-related element-binding factor, DREE Protein Sci 6 ... 

DNA coding for the 21-amino-acid chain and the other carrying the DNA sequence coding for the 30-amino-acid chain. In both plasmids, the DNA sequence was linked to instructions for another protein, an enzyme protein. The bacteria produced two fused proteins of enzyme and one of the two insulin chains. 

PCR and in vitro recombination reactions are quite simple and straightforward for generating multiple expression plasmids in parallel, e.g., in a 96-well plate see Fig. 3a). The first preliminary expression experiment was done to evaluate the production level of each GST-fused protein. In this step, we compared the staining patterns of E. coli proteins harboring expression plasmids with the patterns of proteins harboring empty vectors on sodium dodecyle sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under the same culture conditions see Fig. 3b). In addition to... 

Fig. 3. Fiow diagram of the expression and purification (a) and a representative resuit of the first preiiminary expression experiment (b) of GST-fused recombinant proteins. Fiow diagram (c left) and the representative resuit (c right) of the removai of GroEL directiy binding expressed GST-fused proteins on giutathione sepharose beads. The whoie ceii iysate of expressed bacteria and each wash and eiution fraction are ioaded on 12% SDS-PAGE (the numbers at the top of the panei are identicai to the numbers in the ieft diagram).

Check the production level and compare the predicted molecular weights of each GST-fused protein sec Fig. 3b and Note 3). 

Elute the GST-fused protein by adding 5 mL of glutathione buffer (see Note 6). 

After negative staining, the band corresponding to the GST-fused protein is excised from the gel by using a surgical knife. 

Kapust RB, Waugh DS. (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8, 1668-74. 

The fusion method is very advantageous for downstream processing, especially when the fused protein is produced in soluble form, thus circumventing the need for renaturation. In addition, it allows the insertion of affinity tails or tags by fusion of... 

This chapter describes the construction of an Fab sequence bearing a foreign gene on the truncated antibody heavy chain, and gives a typical protocol for expression of the fused protein in Escherichia coli. 

Shirabe, K., Yubisui, T. Takeshita, M. (1989). Expression of human erythrocyte NADH-cytochrome 65 reductase as a thrombin-cleavable fused protein in Escherichia coli. Biochimica et Biophysica Acta 1008, 189-92. 

Balia, T., and Varnai, P., 2002, Visualizing cellular phosphoinositide pools with GFP-fused protein-modules. Sci. STKE 2002 PL3. 

Fig. 15.3 The fusion protein has the annino-ternninal part of the Ber kinase and the carboxy-ternninal part of the Abl tyrosine kinase. The result is that the Abl kinase in the fused protein is hyperactive and drives proliferation of haematopoietic cells in the bone marrow. (See also Rgs 24-25 of ref. 31, reproduced with permission of Taylor and Francis, Inc.)...

Since Rluc and GEP recombinantly fused proteins can be expressed in living cells, BRET is an interesting tool for monitoring molecular interactions in cell-based assays. BRET has been particularly used for the study of GPCRs by probing receptor oligomerization or activation. ... 

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