Difference between revisions of "Part:BBa K2255003"

Revision as of 22:21, 13 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 [http://2017.igem.org/Team:Aix-Marseille/DEPS enzyme] that use to disrupt the biofilm.

GFP purification with a Ni column

A)Loading sample. B)Wash. 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 are gone with the wash. 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