Difference between revisions of "Part:BBa K844000"

Revision as of 23:23, 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 using Assembly Standard #23 (Silver Fusion); 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 Coomassie stained SDS PAGE gel showing the spider silk protein (~25.4 kDa) that was purified using a Nickel affinity resin column. The protein in the lane is nearly pure, with only minor bands at 24 kDa and 15 kDa as contaminants. The 10x-His tag aids in producing highly pure protein samples as it binds more tightly to the column, allowing higher concentration wash steps to be used to remove contaminants from the sample. Also 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 (~25.4 kDa). This demonstrates that the 10x-histidine tag is functioning and is compatible with staining and antibody techniques used with 6x-histidine tags.

Figure 1. Coomassie blue stained SDS PAGE gel showing highly pure spider silk sample. The first lane contains the cell lysate sample. The second lane is the flowthrough from the column; note the absence of spider silk band from this flowthrough, indicating the high affinity of the 10x-His tag. The third lane is the eluted spider silk band, with only very minor contaminating bands. The last lane is the Biorad Precision Plus Dual Color Protein Standard, with protein sizes indicated in kDa.

Figure 2. 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 attached to the secondary antibody.

Review of Existing Histidine Tags in the Parts Registry

A series of searches of the parts registry were conducted to determine what His tags exist prior to the creation of this part. The vast majority of parts containing His tags were basic parts; most of the these parts likely were synthesized with the His tag or had it added later through PCR or mutagenesis. Very few independent His tags exist in the registry, and below is a brief description of the ones we found and a discussion of how this part differs or improves upon them.

Bba_J70044 - This part is a 6x-His tag with a single stop codon. It contains additional glycine and serine residues upstream of the His tag, and seems to be utilized only in conjunction with the BioScaffold system used by the Knight lab in 2009. The BBa_K844000 10x-His tag improves on this part by extending the His repeat region to 10x, removing the additional glycine and serine residues upstream, and adding an additional stop codon downstream to ensure protein termination. BBa_K844000 is also readily compatible with the Assembly Standard #23 cloning system.

BBa_J18909 - This part

BBa_K112703 BBa_T2000 BBa_549019

J70044 is part of some weird scaffolding system T2000 is only a 7x tag, but has TAATAA - this was one of Reshma Shetty's parts K18909 uses standard #25 K112703 uses standard #21

K549019 is actually an existing 10xHis tag for E. coli using the #10 standard I believe. Interestingly, I decided to translate it and got:

I R G R F Stop Met A G H H H H H H H H H H T G Stop

It is essentially worthless as it has an upstream stop codon in frame with the 10x-His tag, and only a single stop at the very end (with 2 amino acids preceding it as well).

Related Parts

BBa_K844020 - A spider silk protein generator part with this 10x-His tag attached to the C-terminus end. The SDS Page and Western blot figures above were created using BBa_K844020.

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