Part:BBa_K2959004

Expressible Pinus sylvestris Defensin 1

This composite part consists of a lacI regulated promoter, ribosome binding site, a coding sequence for Pinus sylvestris Defensin 1 as a fusion protein with a 6x His-Tag, and a double terminator. This construct allows the expression of PsDef1, an antifungal peptide from Scots pine seeds, in E. coli. Expression can be positively regulated by the addition of IPTG or lactose thanks to the lacl regulated promoter. The part is designed to code for a fusion protein of PsDef1 with a polyhistidine tag (6x His-Tag) at its N-terminus for purification by immobilized metal affinity chromatography.

Usage and Biology

PsDef1 is present in Pinus sylvestris seeds, vegetative and generative organs. The defensin encodes a protein of 83 amino acids in length, whose first 33 amino acids correspond to the ‘N-terminal signal peptide, it also has eight highly conserved cysteines residues that are predicted to form four disulfide bonds C3–C49, C14–C34, C20–C43, and C24–C45¹, which stabilize its structure making it resistant to environmental changes². Its conformation consists in a cysteine-stabilized αβ-motif (CSαβ) with a prominent α-helix and a triple-stranded antiparallel β-sheet that is stabilized by the 4 disulfide bonds¹.

Endogenous PsDef1 causes morphological changes in fungi mycelium when interacting with the sphingolipid membrane on fungal cell, disrupting the membrane integrity. Antifungal activity towards phytopathogenic fungi Botrytis cinerea, Fusarium oxysporum, Fusarium solani, and Heterobasidion annosum has been demonstrated; as well as its antimicrobial activity against Gram-positive (Bacillus pumilus) and Gram-negative bacteria (Pectobacterium carotovorum, Pseudomonas fluorescens)¹.

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]

References

1. Khairutdinov, B. I., Ermakova, E. A., Yusypovych, Y. M., Bessolicina, E. K., Tarasova, N. B., Toporkova, Y. Y., ... & Nesmelova, I. V. (2017). NMR structure, conformational dynamics, and biological activity of PsDef1 defensin from Pinus sylvestris. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1865(8), 1085-1094. doi:10.1016/j.bbapap.2017.05.012
2. Kovalyova, V. A., Gout, I. T., Kyamova, R. G., Filonenko, V. V., & Gout, R. T. (2007). Cloning and analysis of defensin 1 cDNA from Scots pine. Biopolymers and Cell,23(5), 398-404. doi: http://dx.doi.org/10.7124/bc.000779