Gene Cloning

264 Gene Cloning
Box 9.1 Translational Fusions and Protein Tags
One of the great powers of recombinant DNA methods is that they allow
you to construct novel genes that encode proteins which do not occur in
nature. An example of this is the construction of so-called translational
fusions. In a translational fusion the promoter, ribosome binding site, start
codon and part of the coding sequence from one gene are fused in the
same reading frame to all or part of another protein. It is important that
the proteins are fused so that the coding sequences are in the same reading
frame otherwise the second protein will not be translated. When this
new gene is expressed, it will be translated into a polypeptide chain containing
the encoded amino acids from the two original proteins, and
remarkably this will often fold up into a protein that now has properties
derived from both of the starting proteins. Translational fusions have a
range of applications including fusion to reporter proteins for analyzing
gene expression (Section 10.2), adding short stretches of amino acids to
proteins to make them easier to purify, and fusing them with soluble partners
to increase solubility.
Adding “tags” (short stretches of amino acids from one protein) onto the
N-terminal or C-terminal ends of another protein can be useful in a number
of situations. First, it is often the case that you wish to identify a protein
in a complex mixture, but you have no way of picking out that particular
protein. However, if the protein is expressed as a translational fusion
with part of another protein for which you have an antibody available, the
new protein will now be recognized by the antibody. This process is called
“epitope tagging”, an epitope being the name given to the part of the protein
which is recognized by the antibody. A commonly used “tag” is a
domain from a human gene called “myc”, for which there are highly specific
antibodies available, which importantly will not cross-react with
other proteins in an E. coli or yeast cell extract. Second, many proteins are
insoluble when expressed at high levels in a heterologous host such as
E. coli. In many cases, this problem can be solved by tagging the protein
with a domain from a highly soluble protein such as maltose binding protein
(MBP). Finally, tags can be used to help purify the protein. An example
of this is the use of “his” tags. Short stretches of DNA that code for several
histidine residues can be fused to the N or C terminus of the protein of
interest, and this will often enable the protein to be purified in one or a few
steps on a column with a high affinity for histidine residues, as described
in the final section in this chapter.
Often the fusion is constructed in such a way that the fused amino acids
can be cleaved off from the other protein after purification has taken place,
for example with a specific protease. This step, however, can be problematical
as it may be difficult to remove all of the tag amino acids.