Gene Cloning

122 Gene Cloning
The main difficulty with antibody-based technology is that it is necessary
to raise a specific antibody for each protein that you wish to detect.
This usually means being able to purify the specific protein in sufficient
quantity to be able to inject it into a small mammal, usually a mouse, rat or
rabbit, in order to cause an immune response. Serum is then harvested
from the animal and the antibody may be further purified before use. This
is a lengthy and costly procedure and can only be carried out successfully
with proteins which can be produced in reasonably large amounts.
It is common to use vectors derived from bacteriophage λ (Section 4.10)
in the construction of libraries for antibody screening. This is because
when you plate a bacteriophage λ library, plaques are produced where the
bacteria have been lysed (Figure 4.3). This means that proteins are released
from the bacterial cells and are readily available to the antibodies and there
is no need for subsequent lysis of the bacterial colonies.
Q5.3. You want to construct a DNA library, which you intend to screen
with an antibody, for expression of a eukaryotic gene. (a) What kind of DNA
library would be most suitable? Give your reasons. (b) What type of vector
would you construct your library in? Explain your choice.
Preparation of filters
To use immunological screening we need to immobilize the proteins, produced
by each of the phage in our library, on a solid support, in such a way
that we know which original plaque they were produced by. This technique,
often called a “plaque lift”, is outlined in Figure 5.3. For immunological
screening nitrocellulose filters are used, as proteins will bind to
them. The filters are then bathed in a solution containing the primary antibody,
which will bind specifically to the protein it was raised against.
Excess antibody is washed off and the bound primary antibody is detected
using a secondary antibody conjugate (Figure 5.2). Using this technique it
is possible to pinpoint which of the original plaques was produced by a
phage expressing the protein of interest. It is often difficult to be sure
exactly which plaque corresponds to the signal on the filter, especially if the
library was plated at high density. In this case a plug of agar is removed
from the area of the positive signal, phage are grown up from it and
replated at a lower density and the screening process is repeated. Once a
single plaque on an agar plate has been identified, which is expressing the
protein of interest, the phage can be isolated for further study.
Q5.4. Describe the approach you would use to clone the gene for (a) β-
galactosidase (see Section 3.9) from the Gram negative bacterium
Citrobacter, and (b) human insulin.You should consider what type of DNA
library would be the most suitable and what form of screening for expression
you could use.