FEATURES OF EXPRESSION VECTORS
Because foreign protein can be toxic to E. coli, especially if made in large amounts, the promoter used to express the foreign gene is critical.
critical 대단히 중요한
If too much foreign protein is made, the host cell may die. To control protein production, expression vectors have promoters with on/off switches; therefore, the host cell is allowed to grow and then after sufficient amounts of bacteria are produced, the gene of interested is turned on.
One commonly used promoter is a mutant version of the lac promoter, lacUV, which drives a very high level of transcription, but only under induced conditions (Fig. 3.23).
transcription 전사
FIGURE 3.23 Expression Vectors Have Tightly Regulated Promoters
An expression vector contains sequences upstream of the cloned gene that control transcription and translation of the cloned gene. The expression vector shown uses the lacUV promoter, which is very strong, but inducible. To stimulate transcription, the artificial inducer, IPTG, is added. IPTG binds to the LacI repressor protein, which then detaches from the DNA. This allows RNA polymerase to transcribe the gene. Before IPTG is added, the LacI repressor prevents expression of the cloned gene.
It has the following elements: a binding site for RNA polymerase, a binding site for the LacI repressor protein, and a transcription start site. The vector has strong transcription stop sites downstream of the polylinker region.
The vector also has the gene for LacI so that high levels of repressor protein are made, thus keeping the cloned genes repressed. Like all vectors, there is an origin of replication and antibiotic resistance gene for selection in bacteria. When a gene library is cloned behind this promoter, the genes are not expressed due to high levels of LacI repressor.
When an inducer, such as IPTG, is added, LacI is released from the DNA, and RNA polymerase transcribes the cloned gene. Another common promoter in expression vectors is the lambda left promoter, or PL. It has a binding site for the lambda repressor.
The gene of interest or library fragment is not expressed unless the repressor is removed. Rather than using its natural inducer, a mutant version of the repressor has been isolated that releases its binding site at high temperatures. So when the culture is shifted to 42°C, the repressor falls off the DNA, and RNA polymerase transcribes the cloned genes.
Another expression system uses a promoter whose RNA polymerase binding site recognizes only RNA polymerase from the bacteriophage T7. Bacterial RNA polymerase will not transcribe the gene of interest.
This system is designed to work only in bacteria that have the gene for T7 RNA polymerase integrated into the chromosome and under the control of an inducible promoter.
integrated 통합된
Some expression vectors contain a small segment of DNA that encodes a protein tag.These are primarily used when the gene of interest is already cloned, rather than for screening libraries.
The gene of interest must be cloned in frame with the DNA for the protein tag. The tag can be of many varieties, but 6HIS, Myc, and FLAG® tag are three popular forms (Fig. 3.24). 6HIS is a stretch of six histidine residues put at the beginning or end of the protein of interest. The histidines bind strongly to nickel.
FIGURE 3.24 Using Tags to Isolate Proteins
Some expression vectors have DNA sequences that code for short protein tags. The 6HIS tag (A) codes for six histidine residues. When fused in-frame with the coding sequence for the cloned gene, the tag is fused to the protein. The 6HIS tag specifically binds to nickel ions; therefore, binding to a nickel ion column isolates 6HIS-tagged proteins. Additionally, antibodies to the 6HIS tag can also be used to isolate the tagged proteins. Other tags, such as Myc or FLAG® (B), are specific antibody epitopes that work in a similar manner. Myc-tagged or FLAG®-tagged proteins can be isolated or identified by binding to antibodies to Myc or FLAG®, respectively
This allows the tagged protein to be isolated by binding to a column with nickel attached. Myc and FLAG® are epitopes that allow the expressed protein to be purified by binding to the corresponding antibody. The antibodies may be attached to a column, used for a Western blot, or seen in vivo by staining the cells with fluorescently tagged versions of the Myc or FLAG® antibodies. (The histidine tag can also be recognized with a specific antibody, if desired.)
The most important feature of expression vectors is a tightly controlled promoter region. The proteins of the expression library are expressed only under certain conditions, such as presence of an inducer, removal of a repressor, or change in temperature. Small tags can be fused into the protein of interest using expression vectors. These tags allow the protein of interest to be isolated and purified.