278 7.4  Molecular Cloning

The nonnative chemical isopropyl-​β-​d-​thio-​galactoside (IPTG) binds to LacI and in doing

so reduces the Lacl affinity to the promoter, thus causing the operon genes to be expressed.

This effect is used in genetic studies involving controllable gene expression in bacteria. Here,

a gene under investigation desired to be expressed is fused upstream of the lac promoter

region in the lac operon and into a plasmid vector using the molecular cloning methods

described earlier in this chapter. These plasmids are also replicated during normal cell growth

and division and so get passed on to subsequent cell generations.

If IPTG is added to the growth media, it will be ingested by the cells, and the repressing

effects of LacI will be inactivated; thus, the protein of interest will start to be made by the

cells, often at levels far above normal wild type levels, as it is difficult to prevent a large

number of plasmids from being present in each cell. Since IPTG does not have an infinite

binding affinity to LacI, there is still some degree of suppression of protein production, but

also the LacI repressor similarly is not permanently bound to the operator region, and so if

even in the absence of IPTG, a small amount of protein is often produced (this effect is com­

monly described as being due to a leaky plasmid).

In theory, it is possible to cater the IPTG concentration to a desired cellular concentration

of expressed protein. In practice though, the response curve for changes in IPTG concentra­

tion is steeply sigmoidal, the effect is largely all or nothing in response to changes in IPTG

concentration. However, another operon system used for genetics research in E. coli and

other bacteria is the arabinose operon that uses the monosaccharide arabinose as the equiva­

lent repressor binder; here the steepness of the sigmoidal response is less than the IPTG

operon system, which makes it feasible to control the protein output by varying the external

concentration of arabinose.

A valuable technique for degrading the activity of specific expressed proteins from genes

in prokaryotes is degron-​targeted proteolysis. Prokaryotes have a native system for reducing

the concentration level of specific proteins in live cells, which involves their controlled deg­

radation by proteolysis. In the native cell, proteins are first marked for degradation by tagging

them with a short amino acid degradation sequence, or degron. In E. coli, an adaptor protein

called SspB facilitates binding of protein substrates tagged with the SsrA peptide to a pro­

tease called “ClpXP” (pronounced “Clip X P”). ClpXP is an enzyme that specifically leads to

proteolytic degradation of proteins that possess the degron tag.

This system can be utilized synthetically by using molecular cloning techniques to

engineer a foreign ssrA tag onto a specific protein that one wishes to target for degrad­

ation. This modification is then transformed into a modified E. coli cell strain in which

the native gene sspB that encodes for the protein SspB has been deleted. Then, a plasmid

that contains the sspB gene is transformed into this strain such that expression of this

gene is under control on an inducible promoter. For example, this gene might then be

switched “on” by the addition of extracellular arabinose to an arabinose-​inducible pro­

moter, in which case the SsrB protein is manufactured that then results in proteolysis of

the SsrA-​tagged protein.

This is a particularly powerful approach in the case of studying essential proteins. An

essential protein is required for the cell to function, and so deleting the protein would nor­

mally be lethal and no cell population could be grown. However, by using this degron-​tagging

strategy, a cell population can first be grown in the absence of SspB expression, and these cells

are then observed following controlled degradation of the essential protein after arabinose

(or equivalent) induction.

KEY POINT 7.5

Proteolysis is the process of breaking down proteins into shorter peptides. Although

this can be achieved using heat and the application of nonbiological chemical reagents

such as acids and bases, the majority of proteolysis occurs by the chemical catalysis due

to enzymes called proteases, which target specific amino acid sequences for their point

of cleavage of a specific protein.