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Introduction to Tags

(Degradation) tags are genetic additions to the end of a sequence that modify expressed proteins in different ways such as marking the protein for faster degradation (see below).

Tags are also used for binding various particles in solution (making them easier to purify in solution from other proteins in a cell). Famous examples of the latter are the [http://en.wikipedia.org/wiki/His-tag "His tag"] [which binds to nickel (Ni++) or cobalt(Co++) ions attached to a matrix] and the [http://www-users.med.cornell.edu/~jawagne/FLAG-tag.html "FLAG tag"] (which binds to an "AntiFLAG" antibody). Both of these tags are commercially available.

Degradation Tags

These genetic tags mark a protein for degradation, thus decreasing a protein's half-life (see each tag's specifications for their respective half-lives). One of the useful aspects of genetic tags is the ability to detect gene activity in a time-sensitive manner.

Tags can be found on:

Note: Tags cannot currently be used separately as BioBricks because currently all of our Protein-coding parts end in the double stop codon "TAATAA".

Mechanism of Use

Currently all of our tags operate through the use of protein-degrading enzymes (proteases) within the cell. They do so by coding for a sequence of about eleven amino acids at the C-terminus of a protein. This sequence is normally generated in E. coli when a ribosome gets stuck on a broken ("truncated") mRNA. Without a normal termination codon, the ribosome can't detach from the defective mRNA. A special type of RNA known as ssRA ("small stable RNA A") or tmRNA ("transfer-messenger RNA") rescues the ribosome by adding the degradation tag followed by a stop codon. This allows the ribosome to break free and continue functioning. The tagged, incomplete protein gets degraded by the proteases ClpXP or ClpAP. When the BioBrick protein-coding sequence ends in a similar degradation tag, the ClpXP or ClpAP proteases can attack it, resulting in a shorter half-life. No such system is yet known for yeast, however ClpXP has been expressed in yeast successfully, so the standard sSRA system is easily portable.

Although originally the number of amino acids encoding for a ssRA/tmRNA tag was eleven (the first sequence that encoded for destruction via ssRA tagging was AANDENYALAA by [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8584937 Keiler et al.]), a subsequent study by [http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed&pubmedid=9603842 Andersen et al.] tested the efficacy of mutating the last three amino acids of that system. Thus our tags (AAV, ASV, LVA, LAA) are classified by only three amino acids.

Future Plans

Our future plans for tags involve building proteins using BioBricks. To read more on this project, visit Ira Phillip's work on BioBrick modification.

References

  1. Jabri and Nature News. "Tag, you're degraded". 2003. [http://www.nature.com/nsmb/journal/v10/n9/full/nsb0903-676.html article]
  2. Karzai, A.W.. "The SsrA−SmpB system for protein tagging, directed degradation and ribosome rescue". 2000. [http://www.nature.com/nsmb/journal/v7/n6/full/nsb0600_449.html Review article]
  3. Keiler, K.C. et al. "Role of a peptide-tagging system in degradation of proteins synthesized from damaged messenger RNA." 1996. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8584937 Pubmed]
  4. Andersen, J.B. et al. "New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria". 1998. [http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed&pubmedid=9603842 link]