An improved SUMO fusion protein system for effective production of native proteins

Chien Der Lee, Hui Chien Sun, Su Ming Hu, Ching Feng Chiu, Atthachai Homhuan, Shu Mei Liang, Chih Hsiang Leng, Ting Fang Wang

Research output: Contribution to journalArticlepeer-review

102 Citations (Scopus)


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. Published by Cold Spring Harbor Laboratory Press.

Original languageEnglish
Pages (from-to)1241-1248
Number of pages8
JournalProtein Science
Issue number7
Publication statusPublished - Jul 2008
Externally publishedYes


  • Enterovirus
  • Foot-and-mouth disease virus
  • Fusion protein
  • Rad51
  • RecA
  • SUMO

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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