國家衛生研究院 NHRI:Item 3990099045/3221
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    题名: An improved SUMO fusion protein system for effective production of native proteins
    作者: Lee, CD;Sun, HC;Hu, SM;Chiu, CF;Homhuan, A;Liang, SM;Leng, CH;Wang, TF
    贡献者: Vaccine Research and Development Center
    摘要: 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 5′-end and XhoI at the 3′-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 His6-Smt3-X) in which the protein of interest (X) is fused to a hexahistidine (His6)-tagged Smt3. Smt3 is the yeast SUMO protein. His6-Smt3-X was purified by Ni2+ resin. Removal of His6-Smt3 was performed on the Ni2+ resin by an engineered SUMO protease, His6-Ulp1 403-621-His6. Because of its dual His6 tags, His6-Ulp1403-621-His6 exhibits a high affinity for Ni2 resin and associates with Ni2+resin after cleavage reaction. One can carry out both fusion protein purification and SUMO protease cleavage using one Ni2+-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.
    日期: 2008-07
    關聯: Protein Science. 2008 Jul;17(7):1241-1248.
    Link to: http://dx.doi.org/10.1110/ps.035188.108
    JIF/Ranking 2023: http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=NHRI&SrcApp=NHRI_IR&KeyISSN=0961-8368&DestApp=IC2JCR
    Cited Times(WOS): https://www.webofscience.com/wos/woscc/full-record/WOS:000257110700013
    Cited Times(Scopus): http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=46449089671
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