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N-Asp-Asp-Asp-Lys-C.

Using this approach, granulocyte-macrophage colony-stimulating factor (GMCSF)

was cloned in and secreted from yeast, and purified in a single step. GMCSF was

produced in the cell as signal peptide-Flag-gene. The signal sequence used was the

signal sequence for the outer membrane protein OmpA. The Flag-gene protein was

thus secreted into the periplasm, the fusion protein purified, and finally the Flag

sequence removed, as described above.

Glutathione affinity agarose

In this method the protein of interest is expressed as a fusion protein with the enzyme

glutathioneS-transferase. The cell extract is passed through a column of glutathione-

linked agarose beads, where the enzyme binds to the glutathione. Once all unbound protein

has been washed through the column, the fusion protein is eluted by passing reduced

glutathione through the column. Finally, cleavage of the fusion protein is achieved using

human thrombin, which recognises a specific amino acid sequence in the linker region.

Protein A

Protein A binds to the Fc region of the immunoglobulin G (IgG) molecule. The protein

of interest is cloned fused to the protein A gene, and the fusion protein purified by

affinity chromatography on a column of IgG-Sepharose. The bound fusion protein is

then eluted using either high salt or low pH, to disrupt the binding between the IgG

molecule and the protein A–protein fusion product. Protein A is then finally removed

by treatment with 70% (v/v) formic acid for 2 days, which cleaves an acid-labile

Asp-Pro bond in the linker region.

Poly(arginine)

This method requires the addition of a series of arginine residues to the C terminus of

the protein to be purified. This makes the protein highly basic (positively charged at

neutral pH). The cell extract can therefore be fractionated using cation-exchange

chromatography. Bound proteins are sequentially released from the column by apply-

ing a salt gradient, with the poly(Arg)-containing protein, because of its high overall

positive charge, being the last to be eluted. The poly(Arg) tail is then removed by

incubation with the enzyme carboxypeptidase B. Carboxypeptidase B is an exopro-

tease that sequentially removes arginine or lysine residues from the C terminus of

proteins. The arginine residues are therefore sequentially removed from the C terminus,

the removal of amino acid residues stopping when the ‘normal’ (i.e. non-arginine)

C-terminal amino acid residue of the protein is reached.

8.4 Protein structure determination

8.4.1 Relative molecular mass

There are three methods available for determining protein relative molecular mass,Mr,

frequently referred to as molecular weight. The first two described here are quick and easy

methods that will give a value to5–10%. For many purposes one simply needs a rough

328 Protein structure, purification, characterisation and function analysis