title: One-step affinity isolation of recombinant protein using the baculovirus/insect cell expression system.
abstract: We have developed two baculovirus transfer vectors which allow single-step affinity isolation of recombinant proteins after expression in insect cells. Using these vectors, recombinant proteins are synthesized as fusions with glutathione-S-transferase and are amenable to enrichment from a crude insect cell lysate using glutathione affinity agarose. After affinity isolation, glutathione-S-transferase can be cleaved from the recombinant polypeptides of interest at an engineered thrombin cleavage site. We used this approach to successfully isolate glutathione-S-transferase, the human low density lipoprotein receptor, two large polypeptides containing cytoplasmic domains of the cystic fibrosis transmembrane conductance regulator (CFTR), and the full-length CFTR. The approach has potential advantages over prokaryotic overexpression of foreign polypeptides, including: (i) eukaryotic post-translational modification of expressed protein, (ii) increased solubility of recombinant fusion proteins synthesized in insect cells leading to increased affinity yield under mild conditions, and (iii) production of large and/or complex polypeptides which might be difficult to purify from prokaryotic cells. The method also allows enrichment of recombinant protein representing a small fraction (less than 5%) of total insect cell protein produced and provides a general method for eukaryotic protein synthesis and isolation which is independent of the particular protein being expressed.
authors.affiliation: Department of Physiology, University of Alabama, Birmingham 35294.
authors.forename: S
authors.initials: S
authors.lastname: Peng
authors.forename: M
authors.initials: M
authors.lastname: Sommerfelt
authors.forename: J
authors.initials: J
authors.lastname: Logan
authors.forename: Z
authors.initials: Z
authors.lastname: Huang
authors.forename: T
authors.initials: T
authors.lastname: Jilling
authors.forename: K
authors.initials: K
authors.lastname: Kirk
authors.forename: E
authors.initials: E
authors.lastname: Hunter
authors.forename: E
authors.initials: E
authors.lastname: Sorscher
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005822:Genetic Vectors / Q000235:genetics; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009036:Moths; D010766:Phosphorylation; D011233:Precipitin Tests; D015202:Protein Engineering / Q000379:methods*; D011973:Receptors, LDL / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D014162:Transfection
chemical_list: D008565:Membrane Proteins; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005982:Glutathione Transferase