A rapid screening method for cell lines producing singly-tagged recombinant proteins using the “TARGET tag” system

Tabata, Sanae; Nampo, Maiko; Mihara, Emiko; Tamura-Kawakami, Keiko; Fujii, Ikuo; Takagi, Junichi

doi:10.1016/j.jprot.2010.05.012

Cited by 23 publications

(15 citation statements)

References 15 publications

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“…2B). Because the level of full-length reelin secreted from transfected cells was generally low (Ͻ50 g/liter), a recently developed affinity tag sequence was attached at the N terminus to facilitate one-step affinity purification from the culture medium using P20.1 antibody (23,35). Reelin samples thus purified were analyzed by SDS-PAGE under a reducing condition and subjected to silver staining to visualize total proteins.…”

Section: Analysis Of Purified Full-length Reelin-the Critical Involvementioning

confidence: 99%

Functional Importance of Covalent Homodimer of Reelin Protein Linked via Its Central Region

Yasui

Kitago

Beppu

et al. 2011

Journal of Biological Chemistry

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Background: Reelin is a large glycoprotein critical in brain development and functions as a form of multimer. Results: Disulfide-bonded homodimer through Cys 2101 is the functional unit of biologically active reelin protein. Conclusion:An intact higher order architecture of reelin multimer is essential for exerting its full biological activity. Significance: Ultrastructural and biochemical characterization of gigantic reelin protein is crucial for mechanistic understanding of reelin signaling.

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Section: Analysis Of Purified Full-length Reelin-the Critical Involvementioning

confidence: 99%

Functional Importance of Covalent Homodimer of Reelin Protein Linked via Its Central Region

Yasui

Kitago

Beppu

et al. 2011

Journal of Biological Chemistry

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“…The TARGET tag consists of 21 amino acids (YPGQÂ5 + V) and is specifically recognized by the P20.1 antibody (Tabata et al, 2010). DNA fragments encoding full-length Enpp1 and Enpp2 from mouse were amplified by PCR using pCAG-GS-Enpp1 and pCAG-GS-Enpp2 as templates and PrimeSTAR MAX DNA polymerase (Takara Bio Inc.).…”

Section: Constructionmentioning

confidence: 99%

“…DNA fragments encoding full-length Enpp1 and Enpp2 from mouse were amplified by PCR using pCAG-GS-Enpp1 and pCAG-GS-Enpp2 as templates and PrimeSTAR MAX DNA polymerase (Takara Bio Inc.). The PCR products were inserted into the XbaI and KpnI sites of pcDNA3.1 (Invitrogen), which had been modified to contain a C-terminal tobacco etch virus (TEV) protease cleavage site followed by the TARGET tag (referred to as pcD-CW; Tabata et al, 2010). Nucleotides 271-279 of the Enpp1 gene (5 0 -AAAGGCCGC-3 0 ) and nucleotides 175-183 of the Enpp2 gene (5 0 -AAAGGTAGA-3 0 ) were replaced by the NotI site-containing sequence (5 0 -CGCGGCCGC-3 0 ; the NotI site is shown in bold) using a PCR-based method.…”

Section: Constructionmentioning

confidence: 99%

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp1

Kato¹,

Nishimasu²,

Mihara³

et al. 2012

Acta Crystallogr F Struct Biol Cryst Commun

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Enpp1 is an extracellular membrane-bound glycoprotein that regulates bone mineralization by hydrolyzing ATP to generate pyrophosphate. The extracellular region of mouse Enpp1 was expressed in HEK293S GnT1 À cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 3.0 Å resolution. The crystal belonged to space group P3 1 , with unit-cell parameters a = b = 105.3, c = 173.7 Å . A singlewavelength anomalous dispersion (SAD) data set was also collected to 2.7 Å resolution using a selenomethionine-labelled crystal. The experimental phases determined by the SAD method produced an interpretable electron-density map.

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“…The most appropriate peptide tag system can be selected from many available peptide tags, such as FLAG tag, (5) TARGET tag, (6) PA tag, (7–16) and MAP tag, (17–20) among many others. (21–23) These systems sometimes have disadvantages, such as low specificity, low affinity, or difficulty in achieving protein elution.…”

Section: Introductionmentioning

confidence: 99%

Development of RAP Tag, a Novel Tagging System for Protein Detection and Purification

Fujii

Kaneko

Ogasawara

et al. 2017

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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Affinity tag systems, possessing high affinity and specificity, are useful for protein detection and purification. The most suitable tag for a particular purpose should be selected from many available affinity tag systems. In this study, we developed a novel affinity tag called the “RAP tag” system, which comprises a mouse antirat podoplanin monoclonal antibody (clone PMab-2) and the RAP tag (DMVNPGLEDRIE). This system is useful not only for protein detection in Western blotting, flow cytometry, and sandwich enzyme-linked immunosorbent assay, but also for protein purification.

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A rapid screening method for cell lines producing singly-tagged recombinant proteins using the “TARGET tag” system

Cited by 23 publications

References 15 publications

Functional Importance of Covalent Homodimer of Reelin Protein Linked via Its Central Region

Functional Importance of Covalent Homodimer of Reelin Protein Linked via Its Central Region

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp1

Development of RAP Tag, a Novel Tagging System for Protein Detection and Purification

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