Difference between revisions of "Part:BBa K2255003"

Revision as of 12:39, 19 October 2017

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

A)Loading sample. B)Unbound. C)Elution.

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.

References

1. ↑ 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
2. ↑ 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