Molecular studies on plant based cyclotides for protein-protein interaction

Abstract

Bioactive agents like secondary metabolites and peptides are gaining much interest while addressing the issues of agricultural and health threats including pests and pathogens and in drug development. Among the plant bioactive peptides, cyclotides being disulphide rich, stable, resistant and having ability to graft epitopes on it or allow sequence variations in its different loops that are very important regarding biological application and research interests for drug development and delivery. The present study was therefore focused on evaluating the bioactive potential of cyclotide bearing indigenous plants including Viola odorata, Viola tricolor, Viola hybrid, Petunia, Clitoria ternatea, pansy F1, Panicum vigatum, Panicum laxam, Panicum maximum and Hamelia patens. The extracts were prepared in phosphate buffer saline (PBS) and protein extraction buffer (PEB). Ptunia and MCOTI-I both separately showed highest DPPH activity (antioxidant) due toas the activity was reduced significantly after treating with Proteinase K. Hamelia possessed highest reducing power, hemolytic, thromobolytic and antimicrobial activites. Mutagenic responses of the (Ames test) medicinal plants were not significant. DNA demage protection assays were also done on all protein extracts including MCOTI-I. Cyclotide genes were also isolated from the selected plant’s DNA using specific primers. It was found that the chimeric arrangement of cliotide gene (from Clitoria plant) as most attractive with conserved sequences in cyclotide and Albumin-1 domains and little intronic variations. Moreover, for cyclotide-protein interaction studies, Texas Red-DBCO amine dye was synthesized to label MCOTI-I-AziF with p-azidophenylalanine Uaa in origami DE3. Optimizations were first done by using the MCOTI-I and its label form MCOTI-I-Lys-TxRd. The click reaction to bind TxRd-DBCO with MCOTI-I-AziF was finally done in buffered guianidinum HCl at a molar ratio of 1:100 and analysed through LC/MS and MCOTI-I-AziF-TxRd-DBCO was confirmed by mass spectrometry. For binding assay FRET analysis was done with Trypsin-EGFP, saturation was achieved with KD value 29.7 ± 1.08 nM. Optimization of same amount of MCOTI-I inteins (using westernblot) was taken in 3-4 h. The IPTG induction avoided the overnight incubations and unwanted backgrounds. The MCOTI-I a cyclotide was expressed for the first time in yeast cells. Current research opens new understandings towards the bioactive xii potential of cyclotide bearing plants with/without peptide content, genetics of cyclotides genes from indegenous plants, optimizations of expression and in vitro labeling studies of MCOTI-I for optical studies regarding drug development and targeting.