T7rna

Strong promoters are used to express high levels of proteins from cloned genes.

Strong virus promoters, such as lambda or T7 promoters are useful for controlling the expression of cloned genes.

use blue and white screening (see above) place the cloned gene under control of the lac promoter, which lies upstream of the multiple cloning site.

Often, the objective of cloning a gene is to isolate high levels of the encoded protein. Purification of proteins has long been complicated because each protein folds up in an individualized manner and consequently behaves differently. To get around this problem the target protein is often tagged with another peptide that is easy to detect and/or purify. This allows purification and manipulation of many different proteins by the same procedures. Tagging is generally done at the genetic level—that is, an extra segment of DNA that codes for the tag is inserted beside the DNA coding for the target protein. This topic is discussed in detail in Ch. 26, Proteomics. For now it should be remembered that when we discuss expression of the "cloned gene", this will in practice often include extra sequences that specify tags or alter regulation to facilitate later analysis.

It is often helpful to deliberately control or enhance expression of a cloned gene, especially if high levels of the encoded protein are needed. Expression vectors are specifically designed to place the cloned gene under control of a plasmid-borne promoter. In practice the gene under investigation is normally first cloned in a general cloning vector and then transferred to the expression vector.

A variety of expression vectors exist with different promoters. The two basic alternatives are very strong promoters and tightly regulated promoters. Strong promoters are used when high levels of the gene product are required. Tightly regulated promoters are useful in physiological experiments where the effects of gene expression are to be tested under a variety of conditions.

Some promoters are both strong and strictly regulated. These are useful when expressing large amounts of a foreign protein in a bacterial cell. Even if the foreign proteins are not actually toxic, the large amounts produced interfere with bacterial growth. Consequently, the bacteria are allowed to grow for a while before the foreign gene is turned on by addition of inducer. The bacteria then devote themselves to manufacture of the foreign protein.

The lac promoter of E. coli is inducible and certain mutant versions exist that are extremely strong promoters, such as the lacUV promoter. IPTG is an artificial inducer that turns on the lac promoter (see Ch. 9). However, repression by LacI, the lac repres-sor, is leaky—i.e. incomplete. Including the lacI gene on a multicopy cloning vector results in high levels of repressor, which turn off the cloned gene more effectively (Fig. 22.31).

Another strictly regulated promoter is the lambda left promoter, PL. The lambda repressor or cI protein represses this promoter. If host cells contain a temperature sensitive version of the cI gene, such as cI857, then raising the temperature can alleviate repression. At 30°C the repressor is functional but at 42°C the repressor is inactivated.

A third popular method is to place the gene under control by a strong promoter from bacteriophage T7. Such promoters are not recognized at all by bacterial RNA polymerase but only by T7 RNA polymerase. Transcription will only occur in specialized host cells that contain the gene for T7 RNA polymerase. Another regulated promoter, such as the lac promoter, in turn controls the expression of T7 RNA polymerase. Induction of the lac promoter induces synthesis of T7 RNA polymerase, which in turn, transcribes the cloned gene (Fig. 22.32). This provides both strict regulation and highlevel expression.

bacteriophage T7 A bacteriophage that infects E. coli whose promoters are only recognized by its own RNA polymerase cI gene Gene encoding the lambda repressor or cI protein cI protein Lambda repressor protein responsible for maintaining bacteriophage lambda in the lysogenic state expression vector Vector specifically designed to place a cloned gene under control of a plasmid-borne promoter lambda left promoter (PL) One of the promoters repressed by binding of the lambda repressor or cI protein lambda repressor (cI protein) Repressor protein responsible for maintaining bacteriophage lambda in the lysogenic state

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