Prohibitin (PHB) is indispensable for Ras-induced Raf-1 activation, cell migration and growth; however, the exact role of PHB in the molecular pathogenesis of cancer metastasis remains largely unexamined. Here, we found a positive correlation between plasma membrane-associated PHB and the clinical stages of cancer. The level of PHB phosphorylated at threonine 258 (T258) and tyrosine 259 (Y259) in human cancer-cell membranes correlated with the invasiveness of cancer cells. Overexpression of phosphorylated PHB (phospho-PHB) in the lipid-raft domain of the cell membrane enhanced cell migration/invasion through PI3K/Akt and Raf-1/ERK activation. It also enhanced epithelial-mesenchymal transition, matrix metalloproteinase-2 activity and invasiveness of cancer cells in vitro. Immunoprecipitation analysis demonstrated that phospho-PHB associated with Raf-1, Akt and Ras in the membrane and was essential for the activation of Raf-1 signaling by Ras. Mice implanted with cancer cells stably overexpressing PHB in the plasma membrane showed enlarged cervical tumors, enhanced metastasis and shorter survival time compared with mice implanted with cancer cells without PHB overexpression. Dephosphorylation of PHB at T258 by site-directed mutagenesis diminished the in vitro and in vivo effects of PHB. These results suggest that increase in phospho-PHB T258 in the raft domain of the plasma membrane has a role in the Ras-driven activation of PI3K/Akt and Raf-1/ERK-signaling cascades and results in the promotion of cancer metastasis.

Expression of recombinant proteins as fusions with SUMO (small ubiquitin-related modifier) protein has significantly increased the yield of difficult-to-express proteins in Escherichia coli. The benefit of this technique is further enhanced by the availability of naturally occurring SUMO proteases, which remove SUMO from the fusion protein. Here we have improved the exiting SUMO fusion protein approach for effective production of native proteins. First, a sticky-end PCR strategy was applied to design a new SUMO fusion protein vector that allows directional cloning of any target gene using two universal cloning sites (Sfo1 at the 59-end and XhoI at the 39-end). No restriction digestion is required for the target gene PCR product, even the insert target gene contains a SfoI or XhoI restriction site. This vector produces a fusion protein (denoted as His 6 -Smt3-X) in which the protein of interest (X) is fused to a hexahistidine (His 6 )-tagged Smt3. Smt3 is the yeast SUMO protein. His 6 -Smt3-X was purified by Ni 2+ resin. Removal of His 6 -Smt3 was performed on the Ni 2+ resin by an engineered SUMO protease, His 6 -Ulp1 403-621 -His 6 . Because of its dual His 6 tags, His 6 -Ulp1 403-621 -His 6 exhibits a high affinity for Ni 2 resin and associates with Ni 2+ resin after cleavage reaction. One can carry out both fusion protein purification and SUMO protease cleavage using one Ni 2+ -resin column. The eluant contains only the native target protein. Such a one-column protocol is useful in developing a better high-throughput platform. Finally, this new system was shown to be effective for cloning, expression, and rapid purification of several difficult-to-produce authentic proteins.Keywords: fusion protein; SUMO; Rad51; RecA; enterovirus; foot-and-mouth disease virus In humans, many diseases, including cancer, aging, anemia, and developmental disorders arise because of gene mutations that result in aberrant proteins. Therefore, proteins are targets for therapeutic drugs, and protein production for structural and functional analysis is a major task in modern biology and medicine. Protein production is never a simple task because proteins are extremely diverse in their physio-chemical properties, and there is no generic 6 These authors contributed equally to this work. Reprint requests to: Ting-Fang Wang, Institute of Biochemical Sciences, National Taiwan University, Taipei 106, Taiwan; e-mail: tfwang@gate.sinica.edu.tw; fax: 886-2-27889759; or Chih-Hsiang Leng, Vaccine Research and Development Center, National Health Research Center, Miaoli 350, Taiwan; e-mail: leoleng@nhri.org.tw; fax: 886-37-583009.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

Background: Plant viruses can be employed as versatile vectors for the production of vaccines by expressing immunogenic epitopes on the surface of chimeric viral particles. Although several viruses, including tobacco mosaic virus, potato virus X and cowpea mosaic virus, have been developed as vectors, we aimed to develop a new viral vaccine delivery system, a bamboo mosaic virus (BaMV), that would carry larger transgene loads, and generate better immunity in the target animals with fewer adverse environmental effects.