Part:BBa_K2144007
Coding sequence for Defensin with His6 and LPXTG-tag regulated by T7-promoter
Defensin-1
Defensin is an antimicrobial peptide (AMP) that contributes to the innate immune response and is present in both vertebrates and invertebrates. It is highly abundant in tissues involved in the host defence system and its amphipathic character allows it to penetrate and disrupt the integrity of pathogenic microbial membranes for destruction. Honey-bee derived defensin-1 was shown to successfully reduce the viability of biofilm-encapsulated wound pathogens, including P. aeruginosa and our main target, S. aureus [1].
Function
The function of defensin is to lyse the pathogenic cell by permeabilizing the plasma membrane. The surface of defensin has both hydrophobic and hydrophilic regions. Upon binding to the microbial membrane, defensin causes the formation of small channels, disrupting the integrity and increasing permeability of the membrane. This results in efflux (outward movement from cell) of vital components in the cell, thus killing it [2].
Biobrick design
The encoding part of the BioBrick is derived from BBa_K1104301 iGEM13 NYMU-Taipei. The T7 promoter, BBa_K525998 made by the Bielefeld Germany iGEM team 2013, has been inserted to control protein expression under IPTG induction. To regulate the protein expression under T7 promoter control, E. coli containing a T7 polymerase must be used, for instance BL21(DE3).
The inserted LT-sequence contain the gene encoding for a Hisx6-tag which enables the use of IMAC to purify the protein. Furthermore it contains the LPETGG-tag which enables the use of the "match making" enzyme Sortase A. The key feature of Sortase is the specific conjugation reaction it carries out, where the enzyme recognizes a specific amino acid sequence, a so called sorting motif (LPETGG motif in the case of S.aureus) and conjugate this sequence with another unit carrying an oligo glycine motif. A new peptide bond is formed. The Linker in the LT-sequence enables proper folding of the protein.
Characterization
Figure 1. Expected size: 33.7 kDa. Result of the SDS-PAGE run at 200 V, 3.00 A. (1) Protein ladder (7) Spider silk protein with Def-LT (8) No sample (9) Spider silk reference (10) Protein ladder. Unrelated samples on the gel were covered for clarity.
A clear band at the expected size of approximately 33.7 kDa, corresponding to defensin-fused spider silk protein, can be seen in well 7 in Figure 1. A faint band just below that band, at an approximate size of 23-24 kDa is visible and probably corresponds to spider silk protein not fused with defensin. The non-fused spider silk reference in well 9 is not visible, which is most likely due to the very small amount added as the total amount was limited.
Figure 2. Expected size: 23 kDa. Result of the SDS-PAGE run at 200 V, 3.00 A. The well marked "SS" is the one with spider silk protein.
Since the reference in the gel (see Figure 1) is not visible, the spider silk-defensin fusion sample was cross-referenced with another SDS-PAGE made earlier with a non-fused spider silk protein sample (see well labeled "SS" in Figure 2). There, the band corresponding to spider silk protein is clearly at a size of approximately 23 kDa, the expected size of the spider silk protein. From this, the conclusion can be made that conjugation of recombinant produced defensin, with attached His-tag and LPETGG recognition motif, to spider silk protein using Sortase A, is proved to work as expected.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 173
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
[1] Sojka M, Valachova I, Bucekova M, Majtan J. Antibiofilm efficacy of honey and bee-derived defensin-1 on multi-species wound biofilm. J Med Microbiol. April 2016 65: 337-344
[2] Tomas Ganz. Defensins: antimicrobial peptides of innate immunity. Nature Reviews Immunology 3, 710-720 (September 2003)
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