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Optimize your experiments to get the best results. We’ve compiled a detailed knowledge base of the top tips and tricks to meet your research needs.
View the relevant questions below:
An expression vector typically contains the following elements:
Additional elements include:
Please view the table below for a list of common constitutive and inducible promoters used.
Host | Constitutive Promoters | Inducible Promoters | Inducer |
E. coli | Not commonly available | Lac (lactose operon); araBAD (arabinose operon) | IPTG |
Yeast | GAP (glyceraldehyde-3-phosphate dehydrogenase) | AOX1 (alcohol oxidase); GAL1 (galactose biosynthesis) | Methanol |
Insect | Ac5 (actin); OpIE1 & 2, PH (polyhedron) | MT (metallothionein) | Copper |
Mammalian | CMV (Cytomegalovirus), EF-1 (human elongation factor-1), UbC (human ubiquitin C), SV40 (Simian virus 40) | Promoter with TetO2 (tetracycline operator); promoter with GAL4 UAS (yeast GAL4 upstream activating sequence) | Tetracycline or doxycycline; |
No; while transcripts will be made, there is no ribosome-binding site (RBS) or Shine-Dalgarno sequence to initiate translation. Therefore, mRNA will be transcribed in E. coli but the message will not be translated into protein.
A ribosome-binding site (RBS) is a segment of the 5' (upstream) part of an mRNA molecule that binds to the ribosome to position the message correctly for the initiation of translation. The RBS controls the accuracy and efficiency with which the translation of mRNA begins.
In prokaryotic mRNAs, the RBS lies about 7 nucleotides upstream from the start codon (i.e., the first AUG). It is also known as the Shine-Dalgarno sequence, which is composed of the polypurine sequence AGGAGG located just 5' of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3'-end of the 16S rRNA in the 30S ribosomal subunit. The Shine-Dalgarno sequence is required.
Protein synthesis in eukaryotes differs from this model. The 5' end of the mRNA has a modified chemical structure ("cap") recognized by the ribosome, which then binds the mRNA and moves along it ("scans") until it finds the first AUG codon. A characteristic pattern of bases (called a "Kozak sequence") is sometimes found around that codon and assists in positioning the mRNA correctly in a manner reminiscent of the Shine-Dalgarno sequence, but not involving base pairing with the ribosomal RNA. Hence, the Kozak sequence is not a ribosome-binding site, but rather a translation initiation enhancer. The consensus Kozak sequence is G/ANNAUGG, where AUG is the initiation codon. A purine (A/G) in position –3 has a dominant effect; with a pyrimidine (C/T) in position –3, translation becomes more sensitive to changes in positions –1, –2, and +4. Expression levels can be reduced up to 95% when the –3 position is changed from a purine to a pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. Note: Yeast do not follow this rule. The optimal Kozak sequence for Drosophila differs slightly (C/AAAA/CAUG).
ATG is often sufficient for efficient translation initiation, although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a consensus Kozak. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.
An IRES is an internal ribosome entry site that allows for end-independent initiation of translation. Researchers typically include an IRES when cloning two or more genes, and want them to be expressed with the same promoter.
Transcription is stopped by a termination sequence that follows your gene of interest. Some examples of transcriptional terminators follow:
E. coli: T7 terminator
Yeast: AOX and CYC1 terminators
Insect: SV40 terminator
Mammalian: SV40 and BGH
Viral: 3’ LTR
Epitope tags are typically included to allow for easy detection or rapid purification of your gene of interest by fusing the tag with your gene of interest. Epitope tags can be on either the N-terminus or C-terminus of your gene of interest. Here are some considerations to take into account when using an epitope tag:
Here are some basic guidelines to help you select an epitope tag:
Purpose | Description | Examples of tag |
Detection | Well-characterized antibody available against the tag | V5, Xpress, myc, 6XHis, GST, BioEase™, capTEV™, GFP, Lumio™ |
Purification | Resins available to facilitate purification | 6XHis, GST, BioEase™, capTEV™ |
Cleavable | Protease recognition site (TEV, EK) to remove tag after expression to get native protein | Any tag with a protease recognition site following the tag (only on N-terminus) |
The differences in the A, B, and C forms of our vectors are a result of either single base pair addition or deletion in the multiple cloning site of the vector. As a result of these single-base changes, we have generated 3 separate reading frames for each type of vector. This feature will enable cloning a gene of interest in-frame with the C-terminal tag using the restriction enzyme of choice. The three reading frames A, B, and C are provided in three separate tubes.
Please use the table below to review the different characteristics each system offers:
Characteristics | E. coli | Yeast | Insect | Mammalian |
Cell growth | Rapid (30 min) | Rapid (90 min) | Slow (18–24 hr) | Slow (24 hr) |
Complexity of growth media | Minimum | Minimum | Complex | Complex |
Cost of growth media | Low | Low | High | High |
Expression level | High | Low–high | Low–high | Low–moderate |
Extracellular expression | Secretion to periplasm | Secretion to medium | Secretion to medium | Secretion to medium |
Post-translational modification | None | Moderate | High | High |
Protein folding | Refolding usually required | Refolding usually required | Proper folding | Proper folding |
N-linked glycosylation | None | High mannose | Simple, no sialic acid | Complex |
O-linked glycosylation | No | Yes | Yes | Yes |
Phosphorylation | No | Yes | Yes | Yes |
Acetylation | No | Yes | Yes | Yes |
Acylation | No | Yes | Yes | Yes |
Gamma-carboxylation | No | No | No | Yes |
Host Organism | Most Common Application | Advantages | Challenges |
Cell-free |
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Prokaryotic |
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Yeast |
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Insect |
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| More demanding culture conditions |
Mammalian |
| Highest-level protein processing |
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Algae |
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For Research Use Only. Not for use in diagnostic procedures.