Molecular Biotechnology. Bernard R. Glick
boxes is also conserved.
Table 3.2 Promoters commonly used for expression of cloned genes in prokaryotic hosts
A high, constitutive (continuous) level of expression of a cloned gene from a strong, unregulated promoter is often detrimental to the host cell because it creates an energy drain, thereby impairing essential host cell functions and growth (Table 3.3). In addition, all or a portion of the plasmid carrying a constitutively expressed cloned gene may be lost after several division cycles, since cells without a plasmid grow faster and eventually take over the culture. Such plasmid instability is a major problem that may prevent the efficient production of a plasmid-borne gene product on a large scale. To overcome this drawback, it is desirable to use a strong regulatable promoter to control transcription in such a way that a cloned gene is expressed only at a specific stage in the host cell growth cycle and only for a specified duration. The production process is performed in two stages. During the first, or growth, stage, the promoter controlling the transcription of the target gene is turned off, while during the second, or induction, stage, this promoter is turned on.
Table 3.3 Factors that may increase the metabolic burden on a prokaryotic host cell that is expressing high levels of a cloned gene
Widely used, strong, regulatable promoters are those from the E. coli lactose (lac) and tryptophan (trp) operons, and from bacteriophage genes such as the gene 10 promoter from E. coli bacteriophage T7 (Table 3.2). Each of these promoters interacts with regulatory proteins (e.g., repressors or activators), which provide a controllable switch for either turning on or turning off transcription of adjacent cloned genes. The lac and trp promoters are recognized by the major form of the E. coli RNA polymerase holoenzyme. This holoenzyme is formed when a sigma factor protein, in this case the sigma factor RpoD, combines with the core proteins of RNA polymerase. The sigma factor directs the binding of the holoenzyme to promoter regions on the DNA.
The E. coli lac promoter is negatively regulated (turned off) by the LacI repressor protein, which prevents the lac operon from being transcribed in the absence of lactose in the growth medium (Fig. 3.2). Induction (turning on) of the lac promoter is achieved by the addition of either lactose or isopropyl-β-D-thiogalactopyranoside (IPTG), a synthetic inducer, to the medium (Fig. 3.3). In the cell, lactose is converted to allolactose by low levels of β-galactosidase that are synthesized when the system is repressed, before it can act as an inducer. The enzyme β-galactosidase is encoded by the lacZ gene of the lac operon, and it is primarily involved in the cleavage of lactose into glucose and galactose. Both allolactose and IPTG can bind to LacI and prevent it from binding to the lac operator, thereby enabling transcription to occur.
Figure 3.2 Diagrammatic representation of the effects of the concentrations of glucose, lactose, and cAMP in the growth medium on the level of transcription from the E. coli lac promoter. The arrow indicates the direction of transcription. The lac repressor is a tetramer. The cAMP–CAP complex binds to a CAP recognition site (CAP box) on the DNA.
Figure 3.3 Inducers of the lac promoter. (A) Lactose, which must be converted to allolactose to be effective; (B) IPTG.
Transcription from the lac promoter is also positively regulated by the binding of the catabolite activator protein (CAP) (also sometimes referred to as the cyclic AMP [cAMP] repressor protein, or CRP) to a region of the DNA (the CAP box) just upstream of the promoter region (Fig. 3.2). When CAP binds to the CAP box, it increases the affinity of the promoter for RNA polymerase, thereby increasing transcription of the genes downstream from the promoter. The affinity of CAP for its binding site on the DNA is enhanced by its association with cAMP, whose level is high when the amount of glucose in the medium is low. Thus, when inducer (lactose or IPTG) is present and there is no repressor bound to the operator, a high intracellular concentration of cAMP can lead to a high level of transcription of the genes downstream of the lac promoter. In practice, lacUV5, a variant of the lac promoter that contains an altered nucleotide sequence in the −10 region and is a stronger promoter than the wild-type lac promoter, is usually used in plasmid expression vectors. In addition, a mutant form of the lacI gene (lacIq) that produces much higher levels of the lac repressor is often employed to decrease basal (background) levels of transcription (transcriptional leakiness) under noninduced conditions (i.e., transcription of a cloned gene in the absence of inducer).
The promoters of bacteriophage genes drive the production of high levels of viral proteins in a host bacterial cell and are often used in biotechnology applications that require a strong promoter. One such promoter from gene 10 of the E. coli bacteriophage T7 activates very high levels of gene expression. However, this promoter is not recognized by the E. coli RNA polymerase but rather it requires the T7 RNA polymerase for transcription. Therefore, to utilize this promoter for transcription of cloned genes, the T7 RNA polymerase gene is inserted in the E. coli chromosome (Fig. 3.4). To regulate expression of the T7 RNA polymerase gene, it is placed under the control of the E. coli lac promoter and operator. During the growth phase, LacI prevents the synthesis of T7 RNA polymerase and the cloned gene product is not produced. When the culture has reached a suitable cell density, IPTG is added to the medium to induce expression of the T7 RNA polymerase gene. The T7 RNA polymerase binds to the T7 gene 10 promoter and transcribes the cloned gene.
Figure 3.4 Regulation of gene expression controlled by the promoter for gene 10 from bacteriophage T7 (pT7). In the absence of the inducer IPTG, the constitutively produced lac repressor, the product of the lacI gene, which is under the control of the lacI promoter, placI, represses the synthesis of the T7 RNA polymerase that is transcriptionally controlled by the lac operator (olac) and lac promoter (plac). In the absence of T7 RNA polymerase, the target gene, which is under the transcriptional control of the T7 gene 10 promoter (pT7), is not transcribed. When lactose or IPTG is added to the medium, it binds to the lac repressor, thereby preventing it from repressing the transcription of T7 RNA polymerase. In the presence of T7 RNA polymerase, the target gene is transcribed. TT, transcription termination sequence.
Promoters must be chosen carefully, especially for the large-scale production of foreign proteins. Chemical inducers can be costly, toxic, or difficult to remove; thermal induction of promoters may induce the production of heat shock proteins, including proteases; nutrient-responsive promoters limit the types of media that can be used for cell growth and induction; and oxygen-regulated promoters often have significant basal levels of activity as a consequence of the inherent difficulty in precisely controlling dissolved oxygen levels in the growth medium. Promoters that are induced when cells enter the stationary phase of the growth cycle may be useful in the design of expression vectors that are employed for large-scale applications. In addition, E. coli is not necessarily the bacterium of choice for the expression of some foreign proteins. While E. coli promoters can regulate the expression of cloned genes in some other bacteria, different promoters may be required in some host strains.
Increasing Translation Efficiency
Expressing a cloned gene from a strong, regulatable promoter, although essential, may not be sufficient to maximize the yield of the cloned gene product. Other factors, such as the efficiency of translation and the stability of the newly synthesized target protein,