Methods Fusion 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).

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). 

Fig. 12.4. Expression systems involving covalent linkages. Numbers correspond to entries (methods) in Table 12.2 (2) labeling of dehalogenase fusion proteins (i.e., HaloTag) with aliphatic chlorides, (4) labeling of AGT (06-alkylguanine-DNA alkyltransferase) fusion proteins with benzylguanine (BG)...

A well-known use of molecular methods is in the study of chromosomal translocations. Thus, in Philadelphia chromosome (ph1) positive chronic myelogenous leukemia (CML), the C-abl oncogene on chromosome 9 is translocated to a region on chromosome 22 called the breakpoint cluster region, or bcr. This (t9 22) translocation results in production of an abnormal fusion protein... 

Kundu et al.64 used MEKC conditions to assess the purity of two recombinant proteins a cytomegalovirus-CMP-KDO synthetase fusion protein expressed in E. coli and a hepatitis C viral protein expressed in CHO cells. Proteins were prepared in a 10-mM Tris-1% SDS buffer (pH 8.5) and analyzed in a 10-mM borate-100-mM SDS buffer (pH 9.5) in uncoated capillaries. The level of impurities, which varied with the method of protein production, agreed within 5% with results obtained by densitometric scanning of SDS-PAGE gels of the same materials. 

The construction of hybrid proteins containing bacterial CBDs may provide a cheap generic method for enzyme immobilization and/or purification using cellulosic matrices. The CBD can be fused at the amino or carboxyl terminus, as in the parent cellulase, to suit individual applications. We have constructed model fusion proteins using the C. fimi CBDs to demonstrate this potential. 

S Kiessig, J Reissmann, C Rascher, G Kiillertz, A Fischer, F Thunecke. Application of a green fluorescent fusion protein to study protein-protein interactions by electrophoretic methods. Electrophoresis 22 1428—1435, 2001. 

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