Highly purified samples of enzymes are essential for the study of their structure and function. The isolation of an individual enzyme, particularly one present in low concentration, from among the thousands of proteins present in a cell can be extremely difficult. By cloning the gene for the enzyme of interest, it generally is possible to produce large quantities of its encoded protein in Escherichia coli or yeast. However, not all animal proteins can be expressed in their appropriately folded, functionally competent form in microbial cells as these organisms cannot perform certain posttranslational processing tasks specific to higher organisms. In these instances, options include expression of recombinant genes in cultured animal cell systems or by employing the baculovirus expression vector of cultured insect cells.

Recombinant Fusion Proteins Are Purified by Affinity Chromatography

Recombinant DNA technology can also be used to generate proteins specifically modified to render them readily purified by affinity chromatography. The gene of interest is linked to an additional oligonucleotide sequence that encodes a carboxyl or amino terminal extension to the protein of interest. The resulting fusion protein contains a new domain tailored to interact with an appropriately modified affinity support.

One popular approach is to attach an oligonucleotide that encodes six consecutive histidine residues. The expressed “His tag” protein binds to chromatographic supports that contain an immobilized divalent metal ion such as Ni2+ or Cd2+. This approach exploits the ability of these divalent cations to bind His residues. Once bound, contaminating proteins are washed off and the His-tagged enzyme is eluted with buffers containing high concentrations of free histidine or imidazole, which compete with the polyhistidine tails for binding to the immobilized metal ions. Alternatively, the substrate-binding domain of glutathione S-transferase (GST) can serve as a “GST tag.” Figure 1 illustrates the purification of a GST-fusion protein using an affinity support containing bound glutathione.

Fig1. Use of glutathione S-transferase (GST) fusion proteins to purify recombinant proteins. (GSH, glutathione.)

The addition of an N-terminal fusion domain may also help induce proper folding of the remainder of the recombinant polypeptide. Most fusion domains also possess a cleavage site for a highly specific protease such as thrombin in the region that links the two portions of the protein to permit its eventual removal.

Site-Directed Mutagenesis Provides Mechanistic Insights

Once the ability to express a protein from its cloned gene has been established, it is possible to employ site-directed muta genesis to change specific aminoacyl residues by altering their codons. Used in combination with kinetic analyses and x-ray crystallography, this approach facilitates identification of the specific roles of given aminoacyl residues in substrate binding and catalysis. For example, the inference that a particular aminoacyl residue functions as a general acid can be tested by replacing it with an aminoacyl residue incapable of donating a proton.