Part:BBa_K2255003
Multi-Tag (Rfc 25)
This tag provides multiple fixation sites: a Strep-tag [1] and a histidine tag. Between those tag is a TEV protease cutting site [2].
This part was designed with the Freiburg (Rfc25) extension. Thus, it contains the restriction sites NgoMIV and AgeI that are compatible and allow the absence of a start and stop codon, which ease the assembly of multiple protein domain.
Usage and Biology
This part is usefull in our to purify the enzyme that use to disrupt the biofilm. Check out our [http://2017.igem.org/Team:Aix-Marseille/DEPS enzyme design page] to know more about our project.
GFP purification with a Ni column
Purification of [sfGFP] fusionned with this tag (BBa_K2255003), with the [Ni-NTA Spin Kit] (Quiagen). As you can see, mainly sfGFP is present after elution and most of the protein weren't bound. Thus, the his-tag of this part is functionnal.
In our [http://2017.igem.org/Team:Aix-Marseille/DEPS project], we wanted to purify the EPS Depolymerase for its use in our cure. Thank to this tag, we will be able to successfully purify our protein with a single Ni column and this will enable us to use the EPS Depolymerase as we wanted and test its activity.
References
- ↑ Schmidt, Thomas GM; Skerra, Arne (2007). "The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins". Nature Protocols. 2 (6): 1528–35. PMID 17571060. doi:10.1038/nprot.2007.209
- ↑ Parks TD, Leuther KK, Howard ED, Johnston SA, Dougherty WG (February 1994). "Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase". Anal. Biochem. 216 (2): 413–7. PMID 8179197. doi:10.1006/abio.1994.1060
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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