Affinity tails

The methods involved in the production of proteins in microbes are those of gene expression. Several plasmids for expression of proteins having affinity tails at the C- or N-terminus of the protein have been developed. These tails are usefiil in the isolation of recombinant proteins. Most of these vectors are commercially available along with the reagents that are necessary for protein purification. A majority of recombinant proteins that have been attempted have been produced in E. Coli (1). In most cases these recombinant proteins formed aggregates resulting in the formation of inclusion bodies. These inclusion bodies must be denatured and refolded to obtain active protein, and the affinity tails are usefiil in the purification of the protein. Some of the methods described herein involve identification of functional domains in proteins (see also Protein engineering). 

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... 

The junction between the affinity tail and the product protein should be in a flexible region to enable independent folding of two polypeptides and to facihtate the cleavage (Nilsson and Abrahamsen, 1990). Some affinity tails and tags are shown in Table 5.14 and described below. 

Staphylococcal protein A (SPA), with its immunoglobulin binding ability, has been used as an affinity tail for the purification of a human insulin growth factor-1 (lGF-1) fusion. The insertion of an acid-labile Asp-Pro cleavage site at the fusion point allowed the separation of the protein A moiety from lGF-1 (Nilsson et al., 1985). 

Table 5.14 Affinity tails useed for purification of gene products by gene fusion.

Another problem that may arise is that the protein fusion may lead to inactivation of the enzyme. The destabilization of P-galactosidase when using a polyphenylalanine lag is an illustradve example (80). There may also be interference between SH groups on the protein and cysteine groups on the fused affinity tail. 

Originally the technique was restricted to proteins having adventitiously exposed histidyl or thiol side-chain groups that can bind to the Mn(IMAC) site.1,82-87 However, the attachment of polyhistidine affinity tails (His , n = 2 to 6) either as an N-terminal or a C-terminal fusion to a desired protein, affords a better binding site on it than any natural protein.88,89 This allows selective separation of the tagged protein. 

Histidine-rich or polyhistidine affinity tails can be associated with proteins and enzymes by fusing the coding sequence of the former with those of the latter [ 147,148]. Such fusion proteins could be immobilized by taking advantage of the specific binding ability of the affinity peptide to appropriate IMA supports. 

Table 3. Native and affinity-tail bearing enzymes immobilized on metal chelate supports...

Affinity tail Affinity tag or type of chromatography Limitations References... 

Example of the variety of tails which have been used are shown in Table 9-4. Some examples of the use of affinity tails demonstrates the versatility of the technique. Mouse dihydrofolate reductase with a polyhistidine peptide tail was purified by using metal chelate chromatography followed by removal of the affinity peptide with car-boxypeptidase A [38], A short hydrophilic antigenic peptide of eight residues engineered on to the N-terminus of recombinant lymphokines allows them to be purified by using an immobilised monoclonal antibody specific for the first four amino acids of the peptide [39]. 

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