title: Purification of a secreted form of recombinant rabies virus glycoprotein: comparison of two affinity tags.
abstract: Expression of recombinant eukaryotic proteins in transfected mammalian cell lines has become an important approach for the characterization of the structure and function of these proteins. However, it is often difficult to recover and purify the recombinant proteins. Therefore, the use of fusion proteins incorporating epitope or affinity tags has become more widespread. In this paper, we directly compare two affinity tags, the hexahistidyl tag and the biotin peptide mimetic, Strep-tag, for use in purification of a recombinant soluble form of rabies virus glycoprotein secreted by transfected Chinese hamster ovary fibroblasts. The recombinant rabies virus glycoproteins are denoted RGP(WT)T441his and RGP(WT)T443s-tag, respectively. These affinity tags were chosen because the chromatographic matrices (Ni(II)-NTA-agarose and recombinant core streptavidin-agarose, respectively) were readily available and these methods offered the possibility of a one-step purification using mild elution conditions. However, in our hands, neither method allowed for a one-step purification protocol. Nonetheless, it was possible to purify RGP(WT)T441his to homogeneity from crude conditioned medium using a combination of metal-chelate affinity chromatography and immunoaffinity chromatography. In contrast, although the Strep-tag has been useful for purifying recombinant proteins expressed in bacteria, we were not able to effectively purify RGP(WT)T443s-tag from conditioned medium using chromatography on recombinant core streptavidin-agarose.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
authors.forename: B S
authors.initials: BS
authors.lastname: Wojczyk
authors.forename: M
authors.initials: M
authors.lastname: Czerwinski
authors.forename: M M
authors.initials: MM
authors.lastname: Stwora-Wojczyk
authors.forename: D L
authors.initials: DL
authors.lastname: Siegel
authors.forename: W R
authors.initials: WR
authors.lastname: Abrams
authors.forename: W H
authors.initials: WH
authors.lastname: Wunner
authors.forename: S L
authors.initials: SL
authors.lastname: Spitalnik
mesh_terms: D000345:Affinity Labels / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D006023:Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012685:Sepharose / Q000378:metabolism; D017421:Sequence Analysis; D019809:Streptavidin; D014162:Transfection / Q000235:genetics; D014759:Viral Envelope Proteins / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000345:Affinity Labels; D000956:Antigens, Viral; D001426:Bacterial Proteins; D000939:Epitopes; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C064951:glycoprotein G, Rabies virus; D006639:Histidine; D001710:Biotin; D012685:Sepharose; D019809:Streptavidin