Wright et al (1991)



Not determined

DiTullio et al (1992)

1 Tissue plasminogen activator

2 Cystic fibrosis transmembrane conductance regulator

Fusion proteins have also been constructed which enable purification of the recombinant product by immobilized metal affinity chromatography (IMAC) (Smith et a!, 1988; Hochuli et al, 1988; Ljungquist et al, 1989). IMAC is a separations method which utilises the interactions between metal ions and certain amino acid residues in proteins, most notably histidine, but also arginine, tryptophan and cysteine (Porath et al, 1975). An IMAC column contains an immobilized chelating agent which can be loaded with a metal ion. A recombinant protein carrying an added amino acid sequence which is rich in these residues binds strongly to the IMAC column. One of the advantages of this technique is that, unlike ion-exchange chromatography, IMAC is compatible with high concentrations of guanidine HC1 and urea. This method can therefore be applied to the purification of denatured proteins which are expressed as inclusion bodies. In addition, histidine is a relatively rare amino acid in proteins and therefore the introduction of histidine sequences significantly increases the adsorption of the recombinant product to an IMAC column compared to natural proteins.

Hochuli et al (1988) modified the gene for mouse dihydrofolate reductase (DHFR) such that the protein expressed in E. coli carried polyhistidine fusions. The effects of the number of histidine residues on the purification of the recombinant protein by Ni2+-IMAC were investigated. They demonstrated that both purity and yield of 90% could be obtained in a single step by this method, although a (His)6 fusion was required to facilitate adsorption to the column in the presence of guanidine HC1. In the absence of guanidine HC1, increasing the number of histidine residues resulted in a decrease in the recovery of the protein from the column. These data are shown in Table 3 to illustrate the principle of chelating peptide fusions for the purification of recombinant proteins by IMAC.

The highly specific nature of biological interactions is an important aspect of the use of enzymes as industrial catalysts. These specific interactions are also used to purify proteins by means of affinity chromatography. A ligand which binds to the protein of interest can be immobilized in a chromatography column to remove the protein from crude material by specific adsorption (see section 5, Biospecific Separations). Fusion proteins which incorporate well-characterized affinity binding regions enable the highly specific isolation of the recombinant product by affinity chromatography. Germino et al (1983) purified a recombinant regulatory protein for which there was no convenient assay system, by constructing a fusion with P-galactosidase. This enabled purification of the recombinant product by affinity chromatography using a P-galactosidase-specific ligand, and detection of the product using p-galactosidase activity. This approach has subsequently been adapted for the purification of various target proteins. Staphylococcal protein A is another commonly studied protein fusion system for affinity purification of recombinant products (Nilsson et al, 1985; Rondahl et al, 1992). This enables secretion from E. coli cells and purification by specific binding to immunoglobulins.

A commercial kit is now available for the expression of recombinant fusion proteins and their affinity purification (Biometra GmbH, Gottingen, Germany). The cloning vector fuses the amino acid sequence Ser-Ala-Trp-Arg-His-Pro-Glu-Phe-Gly-Gly to the C-terminal of the protein which enables purification using a streptavidin affinity column. This technique was developed by Schmidt & Skerra (1993) who used polymerase chain reaction to generate random nucleotide sequences based around streptavidin binding domains. Libraries of sequences were expressed and screened for binding activity, the above being selected as optimal for affinity purification and for the detection of products by blotting or ELISA. Using this method, a recombinant antibody fragment was expressed in E. coli and isolated from a cell lysate with a purification of 650 using a streptavidin-agarose affinity column and elution with 1 mM iminobiotin.

Table 3

The affinity of polyhistidine-dihydrofolate reductase (DHFR) fusion proteins for Ni2+-IMAC adsorbent in the presence of 50 mM phosphate buffer and 6 M guanidine HC1. Data from Hochuli et al (1988).

Table 3

The affinity of polyhistidine-dihydrofolate reductase (DHFR) fusion proteins for Ni2+-IMAC adsorbent in the presence of 50 mM phosphate buffer and 6 M guanidine HC1. Data from Hochuli et al (1988).

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