Tag

Part:BBa_K1162009

Designed by: Kathleen Miller   Group: iGEM13_Utah_State   (2013-09-15)
Revision as of 03:51, 28 September 2013 by Ryanputman (Talk | contribs)

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10x-Histidine (10x-His) Tag with Met (ATG) For N-terminal purification

Design

This part was designed with Assembly Standard #23 (Silver Fusion) to allow for protein fusions. This 10x His-Tag is an improvement upon BBa_K844000 by removing the double stop codon (TAA TAA) to allow for C-terminal fusion of proteins. Another key characteristic of this part is the presence of a Methionine(atg) at the start of the coding region (N-terminal), which enables the user to fuse proteins downstream and then purify. The promoter-rbs system of choice can be placed in front of this His-Tag for protein expression.

Experience

AtgHTGFP.png
Figure 1. The above image shows a GFP dot image (on parafilm, illuminated by UV light) in which it is clear that the N-terminal 10x His-Tag (BBa_K1162009) method of purification works as desired and adds another important purification system to the registry.
AtgHTGFP_protein_gel.PNG
Figure 2. From the SDS-PAGE gel it can be seen that there is pure GFP (~26.9 kDa) in elution fractions number 2 and 3. The wash fractions do not appear to have any GFP, which indicates that the N-terminal 10x His-Tag is strongly bound to the Nickel resin column during washing steps.

Related parts

BBa_K844000 - this link also provides an extensive review of existing Histidine Tags in the Parts Registry.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
n/a10x-Histidine (10x-His) Tag with Met (ATG) For N-terminal purification