- Reduction of the intracellular concentration of toxic proteins: Some cloned proteins are
toxic to the cell in which they are produced and there is therefore a limit to the amount
of protein the cell will produce before it dies. Protein secretion should prevent cell
death and result in continued production of protein. - To allow post-translational modification of proteins: Most post-translational
modifications of proteins occur as part of the secretory pathway, and these
modifications, for example glycosylation (see Section 8.4.4), are a necessary process
in producing the final protein structure. Since prokaryotic cells do not glycosylate
their proteins, this explains why many proteins have to be expressed in eukaryotic
cells (e.g. yeast) rather than in bacteria. The entry of a protein into a secretory pathway
and its ultimate destination is determined by a short amino acid sequence (signal
sequence) that is usually at the N terminus of the protein. For proteins going to the
membrane or outside the cell the route is via the endoplasmic reticulum and Golgi
apparatus, the signal sequence being cleaved-off by a protease prior to secretion. For
example, humang-interferon has been secreted from the yeastPichia pastorisusing
the protein’s native signal sequence. Also there are a number of well-characterised
yeast signal sequences (e.g. thea-factor signal sequence) that can be used to ensure
secretion of proteins cloned into yeast.
Fusion proteins to aid protein purification
This approach requires an additional gene to be joined to the gene of the protein of
interest such that the protein is produced as a fusion protein (i.e. linked to this second
protein, or tag). As will be seen below, the purpose of this tag is to provide a means
whereby the fusion protein can be selectively removed from the cell extract. The
fusion protein can then be cleaved to release the protein of interest from the tag
protein. Clearly the amino acid sequence of the peptide linkage between tag and
protein has to be carefully designed to allow chemical or enzymatic cleavage of this
sequence. The following are just a few examples of many different types of fusion
proteins that have been used to aid protein purification.Flag™
This is a short hydrophilic amino acid sequence that is attached to the N-terminal end of
the protein, and is designed for purification by immunoaffinity chromatography.Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-ProteinA monoclonal antibody against this Flag sequence is available on an immobilised
support for use in affinity chromatography. The cell extract, which includes the
Flag-labelled protein, is passed through the column where the antibody binds to the
Flag-labelled protein, allowing all other proteins to pass through. This is carried out in
the presence of Ca^2 þ, since the binding of the Flag sequence to the monoclonal
antibody is Ca^2 þdependent. Once all unbound protein has been eluted from the
column, the Flag-linked protein is released by passing EDTA through the column,
which chelates the Ca^2 þ. Finally the Flag sequence is removed by the enzyme
enterokinase, which recognises the following amino acid sequence and cleaves the
C-terminal to the lysine residue:327 8.3 Protein purification