Protein Expression Basics Support—Getting Started

An expression vector typically contains the following elements:

• Gene of interest expression cassette (including promoter-gene-termination or poly(A) signal)
• Antibiotic selection cassette for particular host
• Antibiotic selection cassette for E. coli
• Bacterial origin of replication

Additional elements include:

• Cloning site
• Epitope tags
• Secretion signal (at N-terminus)

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:

1. N-terminal tags may have protease cleavage sites
2. N-terminal tags have RBS/Kozak included
3. Secretion signals are always N-terminal and are automatically cleaved off during Golgi processing
4. Replace the native secretion signal if present in the gene of interest
5. Choose an N-terminal tag when the C-terminus of the protein is important for function
6. Ensure that a stop codon is present at the end of your gene of interest when working with an N-terminal tag
7. If including an N-terminal tag, ensure that your gene is in-frame with the tag
8. Choose a C-terminal tag when the N-terminus of the protein is important for function or if the protein is uncharacterized
9. Make sure to omit the stop codon from your gene of interest for fusion with C-terminal tags; a stop codon is present at the end of the C-terminal tag
10. Make sure to include your start codon (ATG) as well as a RBS/Kozak sequence if needed at the start of your gene of interest when working with C-terminal tags
11. If including a C-terminal tag, ensure that the tag is in-frame with your gene of interest

Here are some basic guidelines to help you select an epitope tag:

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: