Molecular cloning and functional analysis of a recombinant ribosome-inactivating protein (alpha-momorcharin) from Momordica charantia

Abstract

Alpha-momorcharin (α-MC), a member of the ribosome-inactivating protein (RIP) family, has been used not only as antiviral, antimicrobial, and antitumor agents, but also as toxicant to protozoa, insects, and fungi. In this study, we expressed the protein in Escherichia coli Rosetta (DE3) pLysS strain and purified it by nickel-nitrilotriacetic acid affinity chromatography. A total of 85 mg of homogeneous protein was obtained from 1 l culture supernatant of Rosetta (DE3) pLysS, showing a high recovery rate of 73.9%. Protein activity assay indicated that α-MC had both N-glycosidase activity and DNA-nuclease activity, the former releasing RIP diagnostic RNA fragment (Endo's fragment) from rice rRNAs and the latter converting supercoiled circular DNA of plasmid pET-32a(+) into linear conformations in a concentration-dependent manner. Specially, we found that α-MC could inhibit the mycelial growth of Fusarium solani and Fusarium oxysporum with IC(50) values of 6.23 and 4.15 μM, respectively. Results of optical microscopy and transmission electron microscopy demonstrated that α-MC caused extensive septum formation, loss of integrity of the cell wall, separation of the cytoplasm from the cell wall, deformation of cells with irregular budding sites, and apoptosis in F. solani. Moreover, α-MC was active against Pseudomonas aeruginosa with an IC(50) value of 0.59 μM. The α-MC protein carries a high potential for the design of new antifungal drugs or the development of transgenic crops resistant to pathogens.

Antimicrobial Activity and Chemical Composition of Momordica Charantia: A Review

“…M. charantia contains triterpenoids 11,39,47 , saponins 48 , polypeptides 49 , flavonoids 14,50,51 , alkaloids 40,52,53 and sterols 13,[54][55][56] , which are distributed throughout the entire plant. The seed is not edible, it contains extractable oils, mostly a conjugated triene cis-9, trans-11, trans-13 (c9, t11, t13) conjugated isomer of linolenic acid, known as α-elestearic acid (α-ESA).…”

“…62,63 MAP30 and α-momorcharin ( Figure 4) are ribosome inactivating proteins (RIP) and have demonstrated antibacterial and antiviral activities. 49,63 Antimicrobial activity Sankaranarayanan and Jolly (1993) have clinically demonstrated the existence of antimicrobial activity on leaf extracts of M. charantia. This activity of M. charantia is attributed to its content of antimicrobial proteins, seed oil, tannins, triterpenoids, alkaloids, cardiac glycosides and steroids.…”

“…37 Recombinant α-momorcharin inhibits the growth of F. solani, causing deformation of cells with irregular outbreaks, integrity loss of the cell wall, rupture of the fungal cell membrane, DNA fragmentation, in addition to affecting macromolecular synthesis and organelles functions. 49 RK29, the active lectin isolated from ripe fruit and seed, inhibits HIV-1 viral reverse transcriptase. 6 Cucurbitane triterpenoids (kuguacins F-S), pentanorcucurbitacins, octanorcucurbitacin and trinorcucurbitacins exhibit weak in vitro anti-HIV-1 activities.…”

“…In addition, cucurbitane are attributed antidiabetic activity 46 , an effect that could enhance treatments against infections in diabetic foot. On the other hand, studies of its safety and efficacy have been carried out in combination with antimicrobials such as aminoglycosides 90 , with the intention of being able to cope with bacterial resistance as well as a decrease in side effects; Therefore, it is recommended to continue studies on proteins such as α-momorcharin and MAP30 which is located in leaves, stems, fruits and seeds (Table 2), which have demonstrated very good antimicrobial metabolites 49,63 as well as some protein fractions 89 .…”

Heterologous Expression and Purification of Recombinant Crotoxin B, the Phospholipase A2 Subunit of Crotoxin

“…The selection of host cells was based on experiments in which these cells proved to be adequate for synthesis of proteins of vegetal [29], animal [30] or human origin [31]. The planned separation method for the recombinant protein was based on Ni 2+ -affinity, thus a 6xHis-tag was added to the coding sequence of CB (isoform C).…”