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The lac promoter, which is part of the lactose utilization operon, was used for many years as the paradigmatic promoter to drive recombinant gene expression. It is chemically‐inducible by the lactose analogon isopropyl‐β‐D‐thiogalactopyranoside (IPTG) and negatively regulated

by the lac repressor protein LacI. In the absence of IPTG, the repressor LacI binds to the

operator region of the lac operon and thus blocks the transcription of target genes. The

presence of IPTG causes derepression by direct binding of IPTG to the repressor, which leads to the dissociation of LacI from the operator due to conformational changes and subsequent

transcription (Lehninger etal., 1994). Additionally, positive regulation of the lac promoter is

mediated by a catabolite activator protein (CAP), whose activity is dependent on the intracellular cAMP concentration. cAMP activates CAP, which then binds to the lac promoter

supporting its activity (Lehninger etal., 1994).

Synthetic promoters like Ptac and Ptrc were also developed, which consist of the ‐35 region of

Ptrp (induced by tryptophane starvation or addition of β‐indoleacrylic acid) and the ‐10 region

of the lacUV5 promoter (mutated derivative of Plac, which is less sensitive to cellular

concentrations of cAMP) (de Boer etal., 1983). Thus, both Ptac and Ptrc possess consensus ‐35

and ‐10 sequences that lead to approximately 11‐times stronger expression levels compared

to the parental promoter PlacUV5 (Brosius etal., 1985, de Boer etal., 1983). A disadvantage of

lac‐derived promoters is their leakiness. Despite the use of host strains that carry the lac repressor LacI, repression can be improved but leakiness is not completely abolished under

non‐inducing conditions (Baneyx, 1999, Jonasson etal., 2002). This makes them not suitable

for the production of proteins, which are toxic or detrimental to the growth of the host cell. Large‐scale protein production with IPTG‐inducible promoters is widely used for basic research, but it is not appropriate for large‐scale induction of human therapeutic proteins due to the toxicity and high costs of IPTG (Hannig & Makrides, 1998, Makrides, 1996). An

alternative could be the induction of lac‐derived promoters by lactose or the choice of

temperature‐sensitive promoters like lac(TS), which is based on a mutant lacI gene, encoding

80  al., 1988, Hasan & Szybalski, 1995). Other heat‐induced promoters used in E. coli are

bacteriophage lambda‐derived PL (λ) (Bernardetal., 1979) and PR (λ) (Elvinetal., 1990). But

thermal induction could be a disadvantage due to the simultaneous induction of heat‐shock proteins including certain proteases that could lead to enhanced protein degradation (Hannig

& Makrides, 1998, Jonasson etal., 2002).

The pET system is the most popular protein expression platform used in E.coli (commercially

available from Novagen, Madison). It is based on a plasmid‐located bacteriophage T7 promoter fused to the lac operator sequence. This T7lac promoter controls the expression of target genes and is repressed under non‐inducing conditions by LacI. The T7 promoter is specifically recognized by the T7 RNA polymerase. The host cell contains a prophage called DE3, which encodes the gene for the T7 RNA polymerase under the control of the IPTG‐ inducible lacUV5 promoter‐operator sequence. In the presence of IPTG, repression by LacI is

abolished and the T7 RNA polymerase is expressed by the induction of PlacUV5. Simultaneously,

the T7 promoter is derepressed, which allows the transcription of target genes by the synthesized T7 RNA polymerase (Dubendorff & Studier, 1991b, Dubendorff & Studier, 1991a,

Studier & Moffatt, 1986, Studier et al., 1990). Thus, a massive overproduction of target

protein up to 50% of total cellular proteins is achievable (Baneyx, 1999). But nevertheless, this system is also not fully repressed in the absence of IPTG because of increasing cAMP concentrations during the stationary growth phase, which lead to the activation of CAP. The presence of T7 lysozyme can decrease basal expression levels due to inhibition of T7 RNA polymerase by direct binding. Therefore, host cells containing a plasmid‐encoded T7 lysozyme (pLys) are often used as expression hosts (Studier, 1991). This tight repression under non‐inducing conditions increases the tolerance to toxic target proteins.

All so far described Plac‐derived promoters were constructed with the purpose to achieve

high levels of protein production. But a massive overproduction is not always beneficial and often results in the formation of inclusion bodies, which contain aggregated target protein (Wilkinson & Harrison, 1991). The araBAD system is another widely used expression system

in E.coli that was developed to escape this disadvantage (Guzman et al., 1995). Expression

vectors named pBAD were constructed, which contain the PBAD promoter of the arabinose

operon and the regulatory gene araC. AraC regulates the expression from PBAD positively and

negatively and negatively autoregulates its own transcription (Carra & Schleif, 1993, Lobell & Schleif, 1990). Upon induction, the inducer molecule L‐arabinose binds to AraC leading to the

expression of PBAD‐controlled target genes (Guzman et al., 1995). The expression level is

lower compared to Plac‐derived promoters. A comparison of the promoters Ptac and PBAD

resulted in a 2.5 to 4.5 stronger activity of Ptac (Guzman et al., 1995). Therefore, this

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to hyperexpression. Under non‐inducing conditions, PBAD is rapidly repressed showing only

very low levels of basal expression. Since the PBAD promoter is subject to catabolite repression

(Miyadaetal., 1984), glucose‐containing media further prevent background expression. Thus,

this system is also used to produce toxic target proteins (Guzman etal., 1995).