Molecular Biotechnology: Principles and Applications - Glick, B., & Pasternak, J. 2002

Fundamentals of Molecular Biotechnology
Optimization of Gene Expression Cloned in Prokaryotic Systems
Gene Expression Mediated by Strong Regulated Promoters

The primary goal of Gene cloning experiments intended for biotechnology Applications is to determine the optimal conditions for efficient expression in a suitable host Organism. Unfortunately, the mere fact that a given gene has been successfully inserted into a cloning vector does not guarantee its expression. At the same time, for a commercial product to be economically viable, its synthesis must proceed at a sufficiently high level. To achieve efficient expression, numerous specialized vectors have already been engineered; this has involved manipulating a wide array of genetic elements that control METABOLISM/31.html">Transcription and Translation Processes, protein stability, and the secretion of products from the host Cell, among others. Among the molecular-biological properties of expression systems, the following are the most critical: 1) the type of transcription promoter and terminator; 2) the strength of mRNA-ribosome binding; 3) the copy number of the cloned gene and its localization (on a plasmid or within the host cell chromosome); 4) the ultimate subcellular localization of the synthesized product; 5) translation efficiency in the host organism; and 6) product stability within the host cell.

There is no universal strategy for optimizing the Expression of cloned genes. Most such genes possess unique molecular properties, and the optimal expression system for each must be empirically determined on a case-by-case basis. The expression efficiency of any foreign gene also depends heavily on its compatibility with the host organism. Although many prokaryotic and eukaryotic organisms are capable of expressing foreign genes, Escherichia coli remains the primary host used for producing commercially valuable products via Recombinant DNA technology. This is primarily because the genetic, molecular-biological, biochemical, and physiological properties of this microorganism have been studied in exhaustive detail. Furthermore, it offers the most cost-effective and rapid means of producing numerous Proteins. Nevertheless, other host systems—such as B. subtilis, Yeasts, animal Cells, and plants—are employed for expressing certain cloned genes, although the strategies originally developed for E. coli systems are generally applicable to these organisms as well.

An essential prerequisite for the efficient expression of any gene is the presence of a strong, regulatable promoter positioned immediately upstream of the gene. Such a promoter exhibits a high affinity for RNA polymerase, ensuring that adjacent sequences are transcribed efficiently and at a high frequency. The regulatability of the promoter allows both The Cell and the researcher to exert strict control over transcription. The promoter of the well-characterized lac (lactose) Operon of E. coli is widely used for expressing cloned genes. However, other promoters possess properties that are equally useful for expression control. To identify such promoters, random DNA fragments are inserted upstream of a so-called reporter gene—which encodes an easily detectable product but lacks its own promoter (Fig. 6.1). If this insertion results in efficient expression of the reporter gene, it is concluded that the cloned fragment contains a functional promoter. Most reporter genes encode either products that confer Antibiotic Resistance or Enzymes identifiable through relatively straightforward colorimetric assays.

It might seem that the most straightforward way to optimize cloned Gene Expression is to insert the gene into a plasmid under the control of a constitutively active strong promoter. However, continuous expression of a foreign gene can prove lethal to the host cell, as it drains cellular energy resources and disrupts normal metabolism. Furthermore, Plasmids carrying a constitutively expressed gene are frequently lost after a few cell cycles because plasmid-free cells grow faster and eventually outgrow the culture. Plasmid instability is the primary obstacle preventing the industrial-scale production of gene products encoded on plasmids. Overcoming this challenge requires implementing expression control so that the cloned gene is expressed only during a specific phase of the Cell Cycle AND for a defined duration; this necessitates The Use of strong, regulatable promoters. Plasmids specifically engineered for this purpose are termed expression vectors.

Class="center">

Fig. 6.1. Identification of strong regulatable promoters. A promoterless reporter gene is inserted into a plasmid. Chromosomal DNA is digested with the restriction endonuclease Abcl, and the resulting fragments are cloned into the plasmid. Efficient expression of the reporter gene indicates that the cloned fragment contains a functional promoter.

Regulatable promoters

The most widely used strong regulatable promoters include the lac and trp operon promoters of E. coli; the custom-engineered tac promoter, which combines the -10 region of the lac promoter and the -35 region of the trp promoter (located 10 and 35 bp upstream of the Transcription initiation site, respectively); the leftward, or p^L, promoter of bacteriophage λ; and the gene 10 promoter of bacteriophage T7. Each of these promoters interacts with specific repressors that mediate the turning on and off of specific gene transcription. Moreover, all of these promoters are recognized by the E. coli RNA polymerase holoenzyme containing the primary sigma factor, which is present in the cell at significantly higher levels than alternative, minor sigma factors. Consequently, transcription does not stall due to a shortage of free sigma factors.

In the absence of lactose in the growth medium, the E. coli lac promoter remains repressed—that is, it is turned off by a repressor protein that blocks Transcription of the lac operon. Induction, or activation of the lac operon, occurs upon The addition of lactose or isopropyl-β-D-thiogalactopyranoside (IPTG) to the medium. Both of these compounds prevent the repressor from binding to the lac operator, thereby allowing transcription to resume.

Transcription driven by the lac promoter is also regulated by the catabolite activator protein (CAP) (Fig. 6.2). The binding of CAP to the promoter increases its affinity for RNA polymerase, thereby enhancing the transcription of downstream genes. In turn, the affinity of CAP for the promoter is augmented when CAP binds cyclic AMP (cAMP), the intracellular levels of which rise as glucose concentrations in the medium decline. Thus, provided the repressor is not bound to the operator, an increase in intracellular cAMP concentrations in the presence of an inducer can stimulate the transcription of genes controlled by the lac promoter.

Fig. 6.2. Effects of glucose, lactose, and cAMP on transcription regulated by the E. coli lac promoter. The arrow indicates the direction of transcription. (Adapted from Abeles et al., 1992, Biochemistry, p. 383, Jones and Bartlett Publishers, Boston, Mass.)

In practice, plasmid expression vectors frequently employ a variant of the lac promoter known as lacUV5, which features an altered -10 sequence and is stronger than the wild-type lac promoter. Transcription from the tac promoter is similarly repressed by the lac repressor and derepressed by adding lactose or IPTG to the medium.

The trp promoter is turned off by the Tryptophan-trp repressor complex, which binds to the trp operator and prevents transcription of the trp operon. Activation (turning on) of the trp promoter is achieved either by depleting tryptophan from the medium or by adding 3-indoleacrylic acid.

Activity of the p^L promoter is regulated by the cI repressor protein of bacteriophage λ. In practice, however, Regulation of transcription from the p^L promoter typically utilizes a Temperature-sensitive mutant form of the cI repressor, designated the cI₈₅₇ protein. Cells synthesizing this repressor are initially grown at 28–30 °C; under these conditions, the repressor blocks transcription from the p^L promoter. Once the culture reaches the desired density (typically mid-log phase), the temperature is raised to 42 °C, which inactivates the cI₈₅₇ repressor and initiates transcription.

Transcription from the bacteriophage T7 promoter requires its cognate RNA polymerase. To utilize this promoter, the T7 RNA polymerase gene is integrated into the E. coli chromosome as part of a λ prophage under the control of the lac promoter. The cells are then transformed with a plasmid containing the target gene under the control of the T7 promoter, and IPTG is added to the medium. This induces the T7 RNA polymerase gene, leading to the synthesis of T7 RNA polymerase, which in turn drives the transcription and Introduction/27.html">Translation of the cloned gene. Often, more than an hour elapses between the Induction of the T7 RNA polymerase gene and the onset of target gene transcription. A diverse series of plasmids, known as pET vectors, has been developed for transcription using the strong T7 promoter.

The efficiency of repressor inactivation—and consequently the activation of transcription—depends on the ratio between the number of repressor molecules and the copy number of the promoter. If the repressor concentration is excessively high, transcription fails to initiate; conversely, if repressor molecules are too scarce (even if they outnumber promoter copies), basal transcription may occur in the absence of induction. Such promoters are often described as being "leaky." Various strategies have been devised to achieve tight control over these regulatable systems. For example, the repressor gene and its corresponding promoter can be placed on two separate plasmids with differing copy numbers, allowing the desired ratio of repressor molecules to promoter copies to be maintained. Typically, the repressor gene resides on a low-copy-number plasmid (with no more than 8 copies per cell), whereas the promoter is located on a multicopy plasmid (with 30–100 copies per cell). Alternatively, the repressor gene can be integrated as a single copy into the host chromosomal DNA, maintaining a low repressor concentration. In systems utilizing the lac promoter, significantly higher levels of the lac repressor can be achieved by replacing the wild-type lacI gene with its mutant counterpart, lacI^q, which suppresses promoter leakiness—namely, reducing the baseline transcription level of the cloned gene in the absence of an inducer.

Production of large quantities of protein products

To yield large amounts of foreign proteins using recombinant E. coli strains, the plasmid pPLc2833 was engineered. It contains a strong promoter, a selectable marker gene, and a short segment harboring several unique restriction endonuclease sites (a polylinker) located immediately downstream of the promoter. The efficiency of this expression vector in synthesizing foreign proteins in E. coli can be further enhanced by replacing the Replication origin of plasmid pPLc2833 with the corresponding origin from plasmid pKN402. This modification increases the copy number of the modified plasmid 5- to 10-fold at 42 °C (Table 6.1). The resulting plasmid, pCP3, carries the p^L promoter and the β-lactamase gene (conferring ampicillin resistance) derived from pPLc2833, combined with the replication origin from pKN402 (Fig. 6.3). Cells harboring this plasmid are initially cultured at 28 °C and subsequently shifted to 42 °C. At the lower temperature, the cI repressor gene integrated into the E. coli DNA is actively expressed, keeping the p^L promoter switched off while maintaining a standard plasmid copy number (Table 6.1). Raising the temperature inactivates the cI repressor, turning the p^L promoter active and driving up the plasmid copy number. Together, these features render plasmid pCP3 a highly efficient expression vector. When the T4 DNA ligase gene was cloned into the polylinker of pCP3, its product accounted for approximately 20% of the total protein synthesized by E. coli at 42 °C, whereas abundant native E. coli proteins, such as the elongation factor EF-Tu, accounted for roughly 2%.

Table 6.1. Dependence of plasmid copy number on temperature for three expression vectors ¹⁾

¹⁾ Adapted from Remaut et al., 1983, Gene 22: 103–113.

Large-scale systems

When cultivating cultures in small volumes (1 to 5 L), expression is typically induced either by shifting the temperature or by adding a chemical inducer. However, in pilot-scale plants (20–100 L) and industrial bioreactors (>200 L), temperatures cannot be altered instantaneously; doing so requires anywhere from 30 to 60 minutes and demands substantial thermal energy. Both time and energy are costly. Similarly, employing chemical Inducers such as IPTG can be prohibitively expensive, potentially rendering the overall process economically unviable. To overcome certain challenges associated with using the p^L promoter for large-scale protein production, a two-plasmid system was developed. The cI repressor gene was placed under the control of the trp promoter and incorporated into a low-copy-number plasmid (Fig. 6.4), ensuring a modest level of repressor synthesis. The second plasmid carried the cloned gene under the control of the p^L promoter. As illustrated in Fig. 6.4A, in the absence of tryptophan, the trp promoter is activated, synthesizing the cI repressor, which in turn switches off the p^L promoter. Conversely, as shown in Fig. 6.4B, when tryptophan is present, the trp promoter is turned off, repressor synthesis ceases, and the p^L promoter operates at full capacity.

Fig. 6.3. Construction of the pCP3 plasmid. A fragment containing the temperature-sensitive replication initiation site (ori) is excised from the pKN402 plasmid using the HaeII restriction endonuclease and ligated with HaeII fragments 1 and 3 of the pPLc2833 plasmid. Fragment 1 contains the p^L promoter and a polylinker (PL), whereas fragment 3 carries the selective marker gene for ampicillin resistance (Amp^r).

Cultures harboring such two-plasmid systems can be grown on cost-effective media based on molasses or casein hydrolysates, which contain minimal amounts of free tryptophan, and induced for cloned gene expression by supplementing the medium with tryptone. The latter provides sufficient free tryptophan to effectively induce transcription. Trial testing of this system demonstrated that the products of the cloned β-lactamase and citrate synthase genes account for 21% and 24%, respectively, of the total synthesized protein following tryptone-induced transcription. Thus, two-plasmid systems enable the industrial-scale and relatively inexpensive production of protein products using recombinant microorganisms.

Fig. 6.4. A two-plasmid system enabling the control of phage λ p^L promoter activity by regulating cI repressor synthesis via tryptophan. The repressor gene cI together with the tryptophan promoter (p trp) are located on one plasmid, whereas the p^L promoter and the cloned gene reside on the other. Arrows indicate the direction of transcription. A. In the absence of tryptophan in the medium, the cI gene is transcribed and translated; the cI repressor binds to the p^L promoter and blocks transcription of the cloned gene. B. In the presence of tryptophan, the cI gene is repressed, its product is not synthesized, and consequently, the cloned gene is transcribed and translated.

Use of alternative microorganisms for expression

E. coli is not the sole microorganism utilized for the synthesis of foreign proteins. Unfortunately, the genetic and molecular biology properties of most other microorganisms are not as thoroughly characterized. Furthermore, there is no single vector or even promoter-repressor system capable of ensuring optimal expression levels in the cells of all, or even just all Gram-negative, Bacteria. Fortunately, many strategies developed for E. coli are applicable to a wide range of other microorganisms, allowing various promoters to be tested for their transcriptional activity in other Gram-negative bacteria. For instance, in one study, a set of plasmid expression vectors was constructed containing the lac, tac, Nm (neomycin resistance gene), and S1 (Rhizobium meliloti ribosomal protein S1 gene) promoters, and the expression level of the β-lactamase gene under the control of each promoter was determined (Table 6.2). It was found that: 1) these promoters exhibit varying degrees of activity across all tested bacterial systems; 2) the tac promoter is most active in E. coli and significantly less active in other bacteria; and 3) Nm is the second most active promoter in E. coli and the most active in other bacteria. Although promoter regions in all Gram-negative bacteria share a similar nucleotide sequence, this does not imply that the most effective promoter for a given organism will be the one most efficient in E. coli. Nevertheless, E. coli promoters can prove quite suitable for regulating the expression of cloned genes in other Gram-negative bacteria as well.

Numerous attempts have been made to construct a "universal" expression vector for Gram-negative bacteria. Eventually, the following strategy was adopted. A 70 bp DNA fragment derived from one of the terminal inverted repeats of transposon 5 (Tn5), along with its corresponding promoter, was inserted into the polylinker of the low-copy-number, broad-host-range plasmid pRK290, yielding plasmid pAV10 (Fig. 6.5). The cloned Tn5 DNA segment contained two independent yet overlapping promoters, each required for the transcription of one of the key Tn5 genes. Since Tn5 is efficiently expressed in diverse bacteria, its promoters can be utilized to drive the transcription of various genes. To test this, the chloramphenicol acetyltransferase and β-galactosidase genes were inserted into the polylinker immediately downstream of the cloned Tn5 promoters. Efficient expression of these genes was observed in cells of Alcaligenes sp., E. coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas stutzeri, Pseudomonas fluorescens, and Serratia marcescens. Thus, There is a realistic prospect of employing Tn5 promoters to initiate foreign gene transcription in various bacterial cells.

Table 6.2. β-Galactosidase activity in Gram-negative bacteria harboring a plasmid vector with the E. coli lacZ gene and a heterologous promoter¹)

¹⁾ According to Labes et al., 1990, Gene 89: 37–46.

Fig. 6.5. Cloning vector pAV10 (not to scale). The positions of the tetracycline resistance gene (Tet^r), the BglII restriction endonuclease site, the replication initiation site (ori), the promoter (p), and the polylinker (PL) are indicated. Insertion of a cloned gene into the polylinker places it under the control of the Tn5 promoter (p). The arrow indicates the direction of transcription.