Systematic analysis of fusion and affinity tags using human aspartyl-tRNA synthetase expressed in E. coli.
Fusion and affinity tags are popular tools for the expression of mammalian proteins in bacteria. To facilitate the selection of expression approaches, a systematic comparison was performed. We cloned, sequenced, and expressed in Escherichia coli ubiquitin- and SUMO-hDRS fusion proteins with biotin- or 6xHis-tags. The tagging of hDRS with ubiquitin or SUMO was necessary to express properly folded and biologically active enzyme. Similar enhancement of hDRS activity was obtained by fusion to ubiquitin or SUMO. Ubiquitin, SUMO, biotin, and hexahistidine tags did not appreciably interfere with hDRS activity. Fusion proteins were specifically cleaved without altering the N-terminal of hDRS. After cleavage hDRS remained soluble and active with a specific activity comparable to that of the fused protein. Similar activity was observed with biotin- and 6xHis-tagging of hDRS. Higher purity but significantly lower yields of hDRS were obtained using biotin-tagging. Overall we demonstrated ubiquitin and SUMO fusion proteins similarly enhanced the proper folding of hDRS expressed in E. coli. In comparison to previous expressions of hDRS as a GST fusion, ubiquitin, and SUMO fusions provided higher yields and easier purification and cleavage.
Guzzo
Catherine M
CM
Department of Chemistry, Georgetown University, Washington, DC 20057, USA.
Yang
David C H
DC
D000595:Amino Acid Sequence; D001226:Aspartate-tRNA Ligase / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry* / Q000235:genetics; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D025801:Ubiquitin; D001226:Aspartate-tRNA Ligase