title: Expression and purification of soluble E-Syt2: low protein stability impedes tag removal.
abstract: Affinity tags are valuable tools for high-throughput protein isolation in automated screenings or downstream processing approaches and are also widely used in laboratory applications for quick and easy access to many proteins. Here, we describe the preparative purification of soluble extended synaptotagmin 2 (rE-Syt2) at bench scale for basic structural and functional studies. Due to the low protein stability, a classical purification procedure without affinity tag was more powerful than isolation of His((6))-tagged rE-Syt2 and subsequent proteolytic tag-removal. Furthermore, expression analysis of truncated rE-Syt2 variants suggested a concept of interdependent-domain organization in proteins containing multiple C2 domains.
authors.affiliation: Mikrobiologisches Institut-Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen, Wasserturmstrasse 3-5, D-91054 Erlangen, Germany.
authors.forename: Gerhard J
authors.initials: GJ
authors.lastname: Groer
authors.forename: Martin
authors.initials: M
authors.lastname: Haslbeck
authors.forename: André
authors.initials: A
authors.lastname: Gessner
mesh_terms: D002845:Chromatography / Q000379:methods*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D050857:Synaptotagmins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D050857:Synaptotagmins; D006639:Histidine; D013917:Thrombin