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
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