Difference between revisions of "Part:BBa K1162006"

Revision as of 03:19, 28 September 2013

LL-37 antimicrobial peptide from Humans (Homo sapiens)

LL-37 is an antimicrobial peptide (AMP) from the cathelicidin family of antimicrobial peptides, isolated from the Human (Homo sapiens). It is a 37-residue helical peptide found throughout the human body, exhibiting a broad spectrum of antimicrobial activity as well as a variety of immunomodulating effects (Dürr, Ulrich H.N., et. al 2006). It has the distinction of being the first and only cathelicidin member discovered from humans, and is extremely well studied and characterized for this reason. The peptide is produced via epithelial cells as well as leukocytes in places such as the skin, gastrointestinal tract, and the respiratory tract. This AMP is is cleaved from a larger protein, hCAP-18, in a series of modifications to yield its active form (Sorensen, O.E, et. al 2001).

Design

This part was codon optimized for E. coli using the Life Technologies GeneArt ® software program. By design, this part is Assembly Standard #23 (Silver Fusion) and contains a Methionine(atg) residue at the start of the coding region. Furthermore, this LL-37 sequence does not have any stop codons, which allows for composite BioBrick construction.

Improvement on Existing Parts (as of September 2013)

BBa_K245114 (Slovenia iGEM team 2009) - This part lacks a start codon, which would require additional design to be expressed individually. The BBa_K1162006 part improves upon this one by adding a Met(atg) residue at the beginning of the coding region. It is unclear if Slovenia's part has been codon optimized for E. coli, but our new part has been designed with this in mind by Life Technologies (see design).

BBa_K875009 (Trieste iGEM team 2012) - This part, BBa_K875009, uses the Assembly Standard #10 for both the prefix and suffix and therefore cannot be used for protein fusions, such as in cases of secretion, protein purification, or fusion proteins. This part also has a stop codon which further limits what can be added to the C-terminal end (purification tag/secretion tag). The BBa_K1162006 improves upon this part by using Assembly Standard #23 (Silver Fusion) and by removing the stop codon at the end of the coding region. Additionally, the BBa_K1162006 part was codon optimized by Life Technologies (see design).

Experience

Figure 1. The image above of protein purification fractions demonstrates that there is expression of LL-37 and WAM-1 in E. coli through GFP fluorescence (GFP is at the C-terminal end). Unfortunately, there were issues with binding to the nickel affinity column, which will be troubleshooted in future work.

Figure 2. This image is an SDS-PAGE gel of the protein products from a 10L fermentation run for the LL37-spider silk generating construct using E. coli as the host organism. In the elution fraction of the gel image below, we can see a protein band at approximately 55-60 kDa, the size of the LL37-spider silk protein. From this study we have demonstrated that antimicrobial spider silk can be produced in E. coli and scaled-up. Since we also have additional AMP-spider silk constructs (see [http://2013.igem.org/Team:Utah_State/Parts BioBricks] page for a complete list), the next step would be to manufacture more of these using a similar approach to the LL37-spider silk. In future work, optimization of LL37-spider silk production will also take place.

References

Dürr, Ulrich H.N. Sudheendra, U.S. and Ramamoorthy, Ayyalusamy, LL-37, the only human member of the cathelicidin family of antimicrobial peptides (Review). Biochimica et Biophysica Acta 1758 (2006) 1408–1425.

O.E. Sorensen, P. Follin, A.H. Johnsen, J. Calafat, G.S. Tjabringa, P.S. Hiemstra, N. Borregaard, Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3, Blood 97 (2001) 3951–3959.

Sequence and Features