Statistical analysis was performed using the GraphPad Prism version 4.0 software (GraphPad Software Inc., La Jolla, CA, USA). The Student t test was used to investigate significant differences between groups. Values of P,0.05 were considered statistically significant.  Preparation of an antitumor and antivirus agent: chemical modification of α-MMC and MAP30 from Momordica Charantia L. with covalent conjugation of polyethyelene glycol BACKGROUND: Alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) derived from Momordica charantia L. have been confirmed to possess antitumor and antivirus activities due to their RNA-N-glycosidase activity. However, strong immunogenicity and short plasma half-life limit their clinical application. To solve this problem, the two proteins were modified with (mPEG)(2)-Lys-NHS (20 kDa). METHODOLOGY/PRINCIPAL FINDINGS: In this article, a novel purification strategy for the two main type I ribosome-inactivating proteins (RIPs), α-MMC and MAP30, was successfully developed for laboratory-scale preparation. Using this dramatic method, 200 mg of α-MMC and about 120 mg of MAP30 was obtained in only one purification process from 200 g of Momordica charantia seeds. The homogeneity and some other properties of the two proteins were assessed by gradient SDS-PAGE, electrospray ionization quadruple mass spectrometry, and N-terminal sequence analysis as well as Western blot. Two polyethylene glycol (PEG)ylated proteins were synthesized and purified. Homogeneous mono-, di-, or tri-PEGylated proteins were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The analysis of antitumor and antivirus activities indicated that the serial PEGylated RIPs preserved moderate activities on JAR choriocarcinoma cells and herpes simplex virus-1. Furthermore, both PEGylated proteins showed about 60%–70% antitumor and antivirus activities, and at the same time decreased 50%–70% immunogenicity when compared with their unmodified counterparts. CONCLUSION/SIGNIFICANCE: α-MMC and MAP30 obtained from this novel purification strategy can meet the requirement of a large amount of samples for research. Their chemical modification can solve the problem of strong immunogenicity and meanwhile preserve moderate activities. All these findings suggest the potential application of PEGylated α-MMC and PEGylated MAP30 as antitumor and antivirus agents. According to these results, PEGylated RIPs can be constructed with nanomaterials to be a targeting drug that can further decrease immunogenicity and side effects. Through nanotechnology we can make them low-release drugs, which can further prolong their half-life period in the human body. Momordica charantia L. (MC), a Momordica Linn. genus of the family Cucurbitaceae and commonly known as bitter melon, is a traditional medicine plant indigenous to China. 1 The fruit and seed extracts from MC have been used in China for centuries for antivirus, antitumor, and immunopotentiating agent purposes. 2 In recent years, several ribosome-inactivating proteins (RIPs), including momordica anti-HIV protein (MAP30) and α-momorcharin (MMC), β-MMC, and γ-MMC, a group of which belong to the family of single-chain RIPs, were found to have the ability to inhibit protein biosynthesis in tumor cells by catalytic inactivation of the 60S ribosomal subunit. 3, 4 These proteins were also found to be able to inhibit the multiplication of herpes simplex virus-1 (HSV-1), 4, 5 poliovirus I in Hep2 cells, 6 and acquired human immunodeficiency virus type-l (HIV-l). 7 However, the strong immunogenicity, allergic reaction, and short half-life of these proteins have been considered the major barriers for their application as therapeutic agents in vivo. 8, 9 In recent years, researchers have shifted their focus to other technologies. An established technology, polyethylene glycol (PEG) conjugation (PEGylation), can bestow on proteins several benefits, such as increasing plasma half-life, decreasing toxicity, and reducing immunogenicity and antigenicity. 9, 10 The Food and Drug Administration has approved the PEGylated forms of the therapeutic proteins such as uricase, erythropoietin, granulocyte-colony stimulating factor, interferon, adenosine deaminase, asparaginase, and a growth hormone antagonist. Another technology is nanotechnology, which uses nanomaterials for packing potential therapeutic proteins to extend the half-life period or to make them targeting drugs.