Abstract
PEGylation is a well-established and effective strategy to decrease immunogenicity, which can increase the stability and in vivo half-life time. However, the generation of multi-site modified products is inevitable due to the lysine chemistry, which will bring difficulties in subsequent research, such as purification and quantification. Site-specific modification by mPEG-succinimidyl carbonate (mPEG-SC) is a widely used method for N-terminal conjugation. In this study, we used it for site-directed modification on two ribosome-inactivating proteins (RIPs), alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30), from Momordica charantia L. According to the optimization of previous modification conditions, we compared Macro-Cap SP with SP-Sepharose FF chromatography for separating the final mPEGylated RIPs. Two kinds of methods both can obtain homogenous mPEGylated RIPs which were identified by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing electrophoresis (IEF), and matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI-TOF/TOF) analysis. We also used iodine staining method to detect the amount of unmodified PEG. Furthermore, the inhibition activity of both mPEGylated and non-PEGylated RIPs against human lung adenocarcinoma epithelial A549 cells was detected. All of the results suggested that the mPEGylated α-MMC/MAP30 might be potentially developed as new anti-tumor drugs.
Highlights
IntroductionFamily [1,2,3]
Alpha-momorcharin (α-MMC) and momordica anti-HIV protein of 30 kDa (MAP30) are both type I ribosome-inactivating proteins (RIPs) which are the members of the single chain RIP (SCRIP)family [1,2,3]
When 80 mg of α-MMC or MAP30 was used as substrates, 25.34 mg of mPEGylated α-MMC and 43.1 mg of mPEGylated MAP30 can be obtained
Summary
Family [1,2,3]. Both of these two RIPs can act irreversibly on ribosomes by removing the adenine residue from eukaryotic ribosomal 28S RNA [4]. Various medicinal activities of α-MMC and MAP30 were studied, which included anti-tumor activities in vitro and in vivo. Both of them owned broad-spectrum anti-virus activities against HSV-1, HBV, and HIV [5,6,7]. According to these findings, researchers performed plenty of studies in order to adapt them for clinical use.
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