Expression vectors
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Gene expression from foreign promoters. Many proteins are expressed at low levels in vivo. To produce high levels of a protein, it is often useful to clone the gene downstream of a well-characterized, regulated promoter. Inducing transcription from the regulated promoter thus results in elevated expression of the downstream gene product. If the regulated promoter can be turned off tightly, this also provides a method of conditionally depleting the cell of a gene product. A variety of regulated promoters can be used for this purpose. A few examples are described below.
Ptac The tac promoter/operator (PTAC) is one of the most widely used expression systems. Ptac is a strong hybrid promoter composed of the -35 region of the trp promoter and the -10 region of the lacUV5 promoter/operator. Expression of Ptac is repressed by the LacI protein. The lacI^q allele is a promoter mutation that increases the intracellular concentration of LacI repressor, resulting in strong repression of PTAC. Addition of the inducer IPTG inactivates the LacI repressor. Thus, the amount of expression from PTAC is proportional to the concentration of IPTG added: low concentrations of IPTG result in relatively low expression from PTAC and high concentrations of IPTG result in high expression from PTAC. By varying the IPTG concentration the amount of gene product cloned downstream from PTAC can be varied over several orders of magnitude.
Several potential problems must be considered when expressing a cloned gene product from PTAC.
lacI^q should be cloned on the same plasmid as the regulated gene, because if lacI^q is on the chromosome or on another plasmid there may be insufficient LacI protein to fully repress the Ptac promoter in trans.
The cell viability should be measured at different concentrations of IPTG, because excessive overexpression of a DNA-binding protein may causethe protein to accumulate in inclusion bodies (Nilsson and Anderson, 1991) or inhibit cell growth.
Even when fully repressed, there is some residual expression from PTAC. If this leaky expression causes problems, it may be necessary to clone the gene into an alternative expression vector that is more tightly repressed.
PBAD. The promoter for the E. coli arabinose operon (PBAD or PARA) is a useful alternative to PTAC. When a gene is cloned behind the PBAD promoter, expression of the gene is controlled by the AraC activator. Expression from PARA is induced to high levels on media containing arabinose. Moreover, expression from PARA is tightly shut off on media containing glucose but lacking arabinose. (For an example of a pBAD expression system, see the pBAD vectors marketed by Invitrogen.)
Like the arabinose operon, expression of the E. coli rhamnose operon is tightly regulated by an activator. Expression from the rhamnose promoter (PRHA) is induced to high levels by the addition
of rhamnose.
Phage promoters. Another approach that is widely used for protein overexpression is to place a gene under the control of a regulated phage promoter. A gene may be cloned downstream of a tightly regulated phage promoter that is normally transcribed by the host's RNA polymerase. For example, expression of a gene cloned downstream of the lambda PL promoter can be regulated by the cI repressor. Using the temperature sensitive cI857 repressor allows control of gene expression by changing the growth temperature -- at 30 C the cI857 repressor is functional and it turns off expression of the gene, but at 42 C the repressor is inactivated so expression of the gene ensues. Alternatively, the wild-type cI gene can be placed under the control of another regulated promoter such as the PLAC promoter, allowing regulation by the addition of IPTG. (For an example of a lambda PL expression system, see the PL vectors marketed by Invitrogen.)
Alternatively, a gene may be cloned downstream of a phage promoter that relies on a phage encoded RNA polymerase. Many phage produce a specific RNA polymerase that recognizes a promoter sequence which is quite different from E. coli promoter sequences. Three phage-specific RNA polymerase/promoter systems that are commonly used in expression vectors include T7, SP6, and T3. In addition to recognizing unique promoters, these systems result in very high levels of transcription of the downstream gene. Such high-level transcription can be very useful for overproducing gene products cloned behind the phage promoter, but the expression is so high that it is often toxic to the host cell. To avoid potential toxicity the phage RNA polymerase is only induced when the overexpression is desired. For example, the phage RNA polymerase may be itself cloned behind a regulated promoter, or the polymerase may be introduced to the cell on a defective phage. (For example of a T7 expression system, see the pET vectors marketed by Novagen.)
REFERENCES:
Amann E, Brosius J, Ptashne M. 1983. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene 25: 167-178.
Guzman LM, Belin D, Carson MJ, Beckwith J. 1992. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 177: 4121-4130.
Haldimann, A., L. Daniels, B. Wanner. 1998. Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon. J. Bacteriol. 180: 1277-1286.
Nilsson B, Anderson S. 1991. Proper and improper folding of proteins in the cellular environment. Annu Rev Microbiol. 45: 607-635.
Studier, F., and B. Moffatt. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189: 113-130
Expression vectors
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Gene expression from foreign promoters. Many proteins are expressed at low levels in vivo. To produce high levels of a protein, it is often useful to clone the gene downstream of a well-characterized, regulated promoter. Inducing transcription from the regulated promoter thus results in elevated expression of the downstream gene product. If the regulated promoter can be turned off tightly, this also provides a method of conditionally depleting the cell of a gene product. A variety of regulated promoters can be used for this purpose. A few examples are described below.
Ptac The tac promoter/operator (PTAC) is one of the most widely used expression systems. Ptac is a strong hybrid promoter composed of the -35 region of the trp promoter and the -10 region of the lacUV5 promoter/operator. Expression of Ptac is repressed by the LacI protein. The lacI^q allele is a promoter mutation that increases the intracellular concentration of LacI repressor, resulting in strong repression of PTAC. Addition of the inducer IPTG inactivates the LacI repressor. Thus, the amount of expression from PTAC is proportional to the concentration of IPTG added: low concentrations of IPTG result in relatively low expression from PTAC and high concentrations of IPTG result in high expression from PTAC. By varying the IPTG concentration the amount of gene product cloned downstream from PTAC can be varied over several orders of magnitude.
Several potential problems must be considered when expressing a cloned gene product from PTAC.
lacI^q should be cloned on the same plasmid as the regulated gene, because if lacI^q is on the chromosome or on another plasmid there may be insufficient LacI protein to fully repress the Ptac promoter in trans.
The cell viability should be measured at different concentrations of IPTG, because excessive overexpression of a DNA-binding protein may causethe protein to accumulate in inclusion bodies (Nilsson and Anderson, 1991) or inhibit cell growth.
Even when fully repressed, there is some residual expression from PTAC. If this leaky expression causes problems, it may be necessary to clone the gene into an alternative expression vector that is more tightly repressed.
PBAD. The promoter for the E. coli arabinose operon (PBAD or PARA) is a useful alternative to PTAC. When a gene is cloned behind the PBAD promoter, expression of the gene is controlled by the AraC activator. Expression from PARA is induced to high levels on media containing arabinose. Moreover, expression from PARA is tightly shut off on media containing glucose but lacking arabinose. (For an example of a pBAD expression system, see the pBAD vectors marketed by Invitrogen.)
Like the arabinose operon, expression of the E. coli rhamnose operon is tightly regulated by an activator. Expression from the rhamnose promoter (PRHA) is induced to high levels by the addition
of rhamnose.
Phage promoters. Another approach that is widely used for protein overexpression is to place a gene under the control of a regulated phage promoter. A gene may be cloned downstream of a tightly regulated phage promoter that is normally transcribed by the host's RNA polymerase. For example, expression of a gene cloned downstream of the lambda PL promoter can be regulated by the cI repressor. Using the temperature sensitive cI857 repressor allows control of gene expression by changing the growth temperature -- at 30 C the cI857 repressor is functional and it turns off expression of the gene, but at 42 C the repressor is inactivated so expression of the gene ensues. Alternatively, the wild-type cI gene can be placed under the control of another regulated promoter such as the PLAC promoter, allowing regulation by the addition of IPTG. (For an example of a lambda PL expression system, see the PL vectors marketed by Invitrogen.)
Alternatively, a gene may be cloned downstream of a phage promoter that relies on a phage encoded RNA polymerase. Many phage produce a specific RNA polymerase that recognizes a promoter sequence which is quite different from E. coli promoter sequences. Three phage-specific RNA polymerase/promoter systems that are commonly used in expression vectors include T7, SP6, and T3. In addition to recognizing unique promoters, these systems result in very high levels of transcription of the downstream gene. Such high-level transcription can be very useful for overproducing gene products cloned behind the phage promoter, but the expression is so high that it is often toxic to the host cell. To avoid potential toxicity the phage RNA polymerase is only induced when the overexpression is desired. For example, the phage RNA polymerase may be itself cloned behind a regulated promoter, or the polymerase may be introduced to the cell on a defective phage. (For example of a T7 expression system, see the pET vectors marketed by Novagen.)
REFERENCES:
Amann E, Brosius J, Ptashne M. 1983. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene 25: 167-178.
Guzman LM, Belin D, Carson MJ, Beckwith J. 1992. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 177: 4121-4130.
Haldimann, A., L. Daniels, B. Wanner. 1998. Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon. J. Bacteriol. 180: 1277-1286.
Nilsson B, Anderson S. 1991. Proper and improper folding of proteins in the cellular environment. Annu Rev Microbiol. 45: 607-635.
Studier, F., and B. Moffatt. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189: 113-130