A Modified Metal-Ion Affinity Chromatography Procedure for the Purification of Histidine-Tagged Recombinant Proteins Expressed in Drosophila S2 Cells

Lehr, Ruth; Elefante, Louis; Kikly, Kristine; O'Brien, S.; Kirkpatrick, Robert B.

doi:10.1006/prep.2000.1258

Cited by 32 publications

(21 citation statements)

References 16 publications

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“…The secreted EЈ and E1Ј proteins were purified from the culture medium by affinity chromatography on a chelating Sepharose column that interacts with the copper bound to the His tag (30). Proteins were further purified by size exclusion chromatography using a Superdex 200 column.…”

Section: Methodsmentioning

confidence: 99%

Differential Cholesterol Binding by Class II Fusion Proteins Determines Membrane Fusion Properties

Umashankar

Martín

Liao

et al. 2008

J Virol

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The class II fusion proteins of the alphaviruses and flaviviruses mediate virus infection by driving the fusion of the virus membrane with that of the cell. These fusion proteins are triggered by low pH, and their structures are strikingly similar in both the prefusion dimer and the postfusion homotrimer conformations. Here we have compared cholesterol interactions during membrane fusion by these two groups of viruses. Using cholesteroldepleted insect cells, we showed that fusion and infection by the alphaviruses Semliki Forest virus (SFV) and Sindbis virus were strongly promoted by cholesterol, with similar sterol dependence in laboratory and field isolates and in viruses passaged in tissue culture. The E1 fusion protein from SFV bound cholesterol, as detected by labeling with photocholesterol and by cholesterol extraction studies. In contrast, fusion and infection by numerous strains of the flavivirus dengue virus (DV) and by yellow fever virus 17D were cholesterol independent, and the DV fusion protein did not show significant cholesterol binding. SFV E1 is the first virus fusion protein demonstrated to directly bind cholesterol. Taken together, our results reveal important functional differences conferred by the cholesterol-binding properties of class II fusion proteins.

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Section: Methodsmentioning

confidence: 99%

Differential Cholesterol Binding by Class II Fusion Proteins Determines Membrane Fusion Properties

Umashankar

Martín

Liao

et al. 2008

J Virol

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“…Immobilized metal-affinity chromatography (IMAC) was described in 1975 [5] and was first used for protein purification in 1987 [6,7]. Since then it has been applied to purify many proteins from several expression systems including bacteria [8], yeast [9], mammalian cells [10] and insect cells [11]. Selection of the appropriate expression system is important when correct protein folding and posttranslational modification (e.g., glycosylation and phosphorylation) are crucial for proper functioning of the resultant protein.…”

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confidence: 99%

“…Several monoclonal antibodies that bind to His-tags have been reported [11][12][13][14][15], and some are available commercially. There have been further technical developments, such as a His-tag specific BIACORE TM , enabling analysis of His-tagged proteins using the Sensor chip NTA.…”

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confidence: 99%

Variability in the immunodetection of His-tagged recombinant proteins

Debeljak

Feldman

Davis

et al. 2006

Analytical Biochemistry

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Labeling of recombinant proteins with polypeptide fusion partners, or affinity tagging, is a useful method to facilitate subsequent protein purification and detection. Poly-histidine tags (His-tags) are among the most commonly used affinity tags. We report strikingly variable immunodetection of two His-tagged recombinant human erythropoietins (Epo), wild type Epo (Epo wt ) and Epo containing an R103A mutation (Epo R103A ). Both were engineered to contain a C-terminal six residue His-tag. The cDNA constructs were stably transfected into CHO cells and COS-7 cells. Clones from the CHO cell transfections were selected for further characterization and larger-scale protein expression. Three chromatographic steps were utilized to achieve pharmacologically pure Epo. Conditioned media from the Epo-expressing cell lines and protein-containing samples from each step of purification were analyzed by SDS-PAGE and dot blot, using both monoclonal anti-human Epo antibody (AE7A5) and anti-His antibodies. While the successful incorporation of the His-tag into our constructs was confirmed by Epo binding to Ni 2+ -NTA resin and by μLC/MS/MS amino acid sequencing, the levels of immunodetection of His-tagged protein varied markedly depending on the particular anti His-tag antibody used. Such variability in His-tag immunorecognition can lead to critical adverse effects on several analytical methods. KeywordsEpo; erythropoietin; His-tag; protein purification; His-tag specific antibodies; immunodetection Recombinant DNA technology enables the production of large quantities of highly purified and well-characterized proteins. Methods that will facilitate subsequent protein analysis and purification are of major interest during the initial design of the recombinant protein. Both detection and purification are greatly simplified by engineering the DNA construct so that the encoded protein is fused to a readily detectable affinant peptide partner, a method designated "affinity tagging". There is often a preference for selection of a short affinity tag that can be fused to the target gene more easily using the polymerase chain reaction (PCR) [1,2]. Examples † Corresponding author: Phone: +1 (617) 632-9980, Fax: +1 (617) 632-0401 Email: asytkows@bidmc.harvard.edu. * Address to which proofs should be mailed: Arthur J. Sytkowski, MD, Beth Israel Deaconess Medical Center, 330, Brookline Ave., W/ BL 548, Boston, MA 02215, USA Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In the present study, we generated two cDNAs encoding C-terminal His-tagged variants of recombinant human erythropoietin (Epo) a...

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“…The nonlytic process is more efficient for secreted proteins. Therefore, the S2 cell system is suitable for the efficient expression and secretion of functional heterologous eukaryotic gene products [5][6][7][8]. However, despite the advantages of the S2 cell system, simultaneous stable expression of multiple types of foreign proteins might not also be simple because of the size limitation of an expression vector; construction of expression vector that has large foreign DNA inserts is difficult because of its genetic instability [9].…”

Section: Introductionmentioning

confidence: 99%

Statistical Determination of Optimal Baculovirus Infection Condition for Recombinant Protein Production in Drosophila S2 Cells

Cho

Kim

et al. 2007

Appl Biochem Biotechnol

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Novel bisphenol A (BPA)-degrading bacterial strains, designated as BP-2CK, BP-21DK, and BP-22DK, were isolated from kimchi, a traditionally fermented food. These isolates were identified as Bacillus pumilus and efficiently degraded BPA in a medium supplemented with nutrients such as peptone, beef extract, and yeast extract. Strains BP-2CK, BP-21DK, and BP-22DK successfully degraded 25, 25, and 50 ppm of BPA, respectively, and all strains exhibited BPA-degrading activity in the presence of 10% NaCl. Accumulation of the metabolites including 4-hydroxyacetophenone, one of the intermediates produced by the other BPA-degrading bacteria, was not observed in BPA degradation by the isolated strains. These results indicate that the isolated food-derived bacteria are applicable for the construction of efficient and safer systems for the removal of BPA.

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A Modified Metal-Ion Affinity Chromatography Procedure for the Purification of Histidine-Tagged Recombinant Proteins Expressed in Drosophila S2 Cells

Cited by 32 publications

References 16 publications

Differential Cholesterol Binding by Class II Fusion Proteins Determines Membrane Fusion Properties

Differential Cholesterol Binding by Class II Fusion Proteins Determines Membrane Fusion Properties

Variability in the immunodetection of His-tagged recombinant proteins

Statistical Determination of Optimal Baculovirus Infection Condition for Recombinant Protein Production in Drosophila S2 Cells

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