title: Improved expression and purification of sigma 1 receptor fused to maltose binding protein by alteration of linker sequence.
abstract: Sigma 1 receptor (S1R) is a eukaryotic membrane protein that functions as an inter-organelle signaling modulator and chaperone. Here we report an improved expression of S1R in Escherichia coli as a fusion to maltose binding protein (MBP) and a high-yield purification. Variants with linking amino acid sequences consisting of 0-5 alanine residues between MBP and S1R were created and tested in several E. coli expression strains in order to determine the best combination of construct and host for production of active MBP-S1R. Among the linker variations, the protein containing a 4-Ala linker exhibited superior expression characteristics (MBP-4A-S1R); this construct was most productively paired with E. coli B834-pRARE2 and a chemically defined growth and expression medium. A 3-step purification was developed, including extraction from the E. coli membrane fraction using a mixture of Triton X-100 and n-dodecyl-beta-D-maltopyranoside identified by screening constrainted by retention of binding function, and purification by amylose affinity and gel filtration chromatographies. This procedure yields ∼3.5mg of purified fusion protein per L of bacterial culture medium. Purified MBP-4A-S1R showed a 175-fold purification from the starting cellular lysate with respect to specific ligand binding activity, and is stable during concentration and freeze-thaw cycling.
authors.affiliation: Transmembrane Protein Center TMPC, Center for Eukaryotic Structural Genomics, Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Katarzyna A
authors.initials: KA
authors.lastname: Gromek
authors.forename: Hannah R
authors.initials: HR
authors.lastname: Meddaugh
authors.forename: Russell L
authors.initials: RL
authors.lastname: Wrobel
authors.forename: Fabian P
authors.initials: FP
authors.lastname: Suchy
authors.forename: Craig A
authors.initials: CA
authors.lastname: Bingman
authors.forename: John G
authors.initials: JG
authors.lastname: Primm
authors.forename: Brian G
authors.initials: BG
authors.lastname: Fox
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D006168:Guinea Pigs; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017480:Receptors, sigma / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000097605:Sigma-1 Receptor
chemical_list: D058619:Maltose-Binding Proteins; D017480:Receptors, sigma; D011993:Recombinant Fusion Proteins; D000097605:Sigma-1 Receptor
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