Part:BBa_K3757005

c_KR-12_1

Cyclotide MCoTI-II with Trypsin inhibitor activity from Momordica cochinchinensis. The antimicrobial fragment KR-12 of the human cathelicidin LL-37 is grafted into loop 1 of the cyclotide, a His6-tag is grafted into loop 5. This part can be cloned into Oak1 precursor and co-expressed with CtAEP1 to yield as a cyclic peptide in Nicotiana benthamiana.

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]

Usage and Biology

MCoTI-II is a squash trypsin inhibitor cyclotide, also known as cyclic knottins, and originates from the plant species Momordica cochinchinensis¹. Cyclotides are a class of plant cyclic peptides with a length of around 30 amino acids (aa). They comprise three disulfide bridges, which together with their cyclic backbone form the characteristic cyclic cystine knot motif making the cyclotide structure very rigid. This motif is responsible for cyclotides’ exceptional stability towards proteases and heat. The regions between their six cysteine residues are called loops 1 to 6. Cyclotides are classified into three categories by their sequence: Möbius, bracelet and trypsin inhibitor cyclotides with the latter having a low sequence conservation compared to the other two classes.² In plants, these peptides are expressed as precursors, which are finally localized to the vacuole. There, the actual cyclotide sequences are recognized by specialized cyclizing asparaginyl endopeptidases (AEPs), which are catalyzing the backbone cyclization.³ MCoTI-II itself is 34 aa in length, with the sequence GGVCPKILKKCRRDSDCPGACICRGNGYCGSGSD, and is folded into a cystine stabilized triple-stranded beta-sheet. Its natural function as a trypsin inhibitor is based on its inhibitory loop 1. Thereby, it acts as a defensive agent against the plant’s predators.¹ MCoTI-II does not disrupt cell membranes and does not display antibacterial, nor hemolytic activity.⁴

Cyclotides like MCoTI-II have been used for grafting approaches in the past. Thereby, grafting means the insertion into or the replacement of one of the cyclotide’s loops with another peptide sequence (figure 1). This enables combining the cyclotide’s stability with the inserted peptide’s biological activity⁴. Regarding MCoTI-II, successful grafting into loops 1, 3, 5 and 6 has been demonstrated⁵.

Figure 1: Illustration of the grafting approach. One or multiple loops of the cyclotide MCoTI-II are replaced by another peptide sequence.

This biological part is a grafted construct of MCoTI-II designed and tested by iGEM team Tuebingen 2021. Two different antimicrobial peptides (AMPs) were used and either grafted into loop 1 or loop 6 of the cyclotide. In addition, a His6-tag (BBa_M50428) was grafted into loop 5 for affinity purification and immunodetection. Furthermore, flexible GS-linkers were included at the interface of the cyclotide’s native sequence and the grafted peptide. For further information on the project, please visit project description wiki page of team Tuebingen 2021. The different grafted constructs used by iGEM Tuebingen are listed in table 1.

Table 1: The grafted cyclotide constructs iGEM team Tuebingen 2021 worked with in the wetlab; the vectors in which the constructs were cloned are shown as well.
BBa_K3757002	c_blank	MCoTI-II loop 5 His6	X
BBa_K3757003	c_CHEN_1	MCoTI-II loop 1 CHEN, loop 5 His6	X	X
BBa_K3757004	c_CHEN_6	MCoTI-II loop 6 CHEN, loop 5 His6	X	X
BBa_K3757005	c_KR-12_1	MCoTI-II loop 1 KR-12, loop 5 His6	X
BBa_K3757006	c_KR-12_6	MCoTI-II loop 6 KR-12, loop 5 His6	X

This construct c_KR-12_1 is MCoTI-II with the AMP KR-12 grafted into loop 1, as well as a His6-tag grafted into loop 5. Its sequence and peptide segments are displayed in figure 2.

Figure 2: Peptide sequence and features of the cyclotide grafting construct c_KR-12_1.

KR-12 is the smallest antibacterial fragment of the human cathelicidin LL-37 (BBa_K245114), which has been used by many iGEM teams in the past. LL-37 is an AMP with 37 aa and an alpha-helical secondary structure. It has potent immunomodulatory and antimicrobial properties and can promote wound healing. Furthermore, LL-37 displays cytotoxicity in the micromolar range. The 12 aa long fragment KR-12 is located nearby LL-37’s C-terminus and has the sequence KRIVQRIKDFLR. It also folds into an amphipathic alpha-helix with a cationic and a hydrophobic face. This structural feature is necessary for the peptide’s mode of action of cell membrane disruption by the carpet-model. KR-12 was demonstrated to be a potent antibacterial agent with minimal inhibitory concentrations (MIC) of 2.5 μM to 10 μM against different clinically relevant gram-positive and gram-negative bacterial species. In addition, it does not exhibit hemolytic activity up to a concentration of 80 μM.6

The grafted construct was cloned into an Oak1 precursor peptide (BBa_K3757007) and expressed in N. benthamiana, regulated by a CaMV 35S promoter (BBa_K788000) and a 35S terminator (BBa_K1159307), C-terminally tagged with a c-myc-tag (No part name specified with partinfo tag.). This is summarized as the composite part BBa_K3757007. This composite part was cloned into a vector with the genes encoding CtAEP1 (BBa_K3757001) and GFP (BBa_K3669012) as a reporter gene, forming a 3in1 vector (No part name specified with partinfo tag.). The resulting vector can then be used for transient transfection of N. benthamiana leaves to express the stabilized AMPs. For further information, visit experiments wiki page of team Tuebingen 2021.