Difference between revisions of "Part:BBa K844000"

Revision as of 19:03, 2 October 2012

10x-Histidine (10x-His) Tag with double stop codon (TAATAA)

10x-Histidine tag with double stop codon TAATAA to allow for better extraction and purity of tagged products and protein termination in a single part.

Usage and Biology

This part is a 10x-Histidine tag with two stop codons on the 3’ end. It is intended to be cloned downstream of a protein or protein subunit lacking a stop codon; the 10x-Histidine tag can then be used to extract the protein using Nickel-based affinity columns. Proteins on gels or in cells can also be analyzed with His-tag specific antibodies or dyes.

The 10x-His tag conveys increased binding affinity for nickel resin compared to traditional 6x His tags as shown in Lee and Kim (2009). Their study also noted that the overall purity of the tagged protein eluted from Nickel resin columns also increased with 10x-His tags compared to 6x-His tags.

This part is also an improvement over existing histidine tags as it includes the standard BioBrick double stop codon (TAATAA) after the 10x-His tag, avoiding the need for an additional cloning or mutagenesis step to add in stop codon sequence.

IMPORTANT NOTE: Any dyes, antibodies, affinity resins, etc. designed for use with 6x-Histidine tags will work with this 10x-Histidine tag, and in most cases should work better.

Experience and Results

This 10x-Histidine tag was used to isolate and analyze spider silk protein produced by the 2012 Utah_State iGEM Team. Below is a Western blot utilizing an antibody that binds specifically to histidine tags, showing a spider silk protein band at the correct molecular weight (~21 kDa). This demonstrates that the 10x-histidine tag is functioning and is compatible with techniques used with 6x-histidine tags.

Figure 1. Western Blot of Spider Silk Protein. Protein expressed is BBa_K844020. Control lane is E. coli DH5a cells without spider silk construct. Marker lane is Biorad Precision Plus Dual Color Protein Standard. Staining was done with alkaline phosphatase gene attached to the secondary antibody.

References

Lee J, and Kim S-H. 2009. High-throughput T7 LIC vector for introducing C-terminal poly-histidine tags with variable lengths without extra sequences. Protein expression and purification 63:58–61.

Sequence and Features