Skip to main content

Purification of Recombinant 2XMBP Tagged Human Proteins from Human Cells

  • Protocol
  • First Online:
Cancer Genomics and Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1176))

Abstract

The ability to purify an intact, functional protein or protein complex is an essential step in biochemical characterization studies. Challenges in purification arise when proteins are of low abundance or are unstable resulting in low yields or poor in vitro activity. In this protocol we describe a method to purify active, recombinant human proteins fused to a tandem MBP tag after expression in human 293T cells.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Goeddel DV (1990) Systems for heterologous gene expression. Meth Enzymol 185:3–7

    Article  CAS  PubMed  Google Scholar 

  2. Brondyk WH (2009) Selecting an appropriate method for expressing a recombinant protein. Meth Enzymol 463:131–147

    Article  CAS  PubMed  Google Scholar 

  3. Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60(5):523–533

    Article  CAS  PubMed  Google Scholar 

  4. Jensen RB, Carreira A, Kowalczykowski SC (2010) Purified human BRCA2 stimulates RAD51-mediated recombination. Nature 467(7316):678–683

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Charlton A, Zachariou M (2011) Tag removal by site-specific cleavage of recombinant fusion proteins. Methods Mol Biol 681:349–367

    Article  CAS  PubMed  Google Scholar 

  6. di Guan C, Li P, Riggs PD, Inouye H (1988) Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. Gene 67(1):21–30

    Article  PubMed  Google Scholar 

  7. Kozak M (1987) An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 15(20):8125–8148

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Leong LE (1999) The use of recombinant fusion proteases in the affinity purification of recombinant proteins. Mol Biotechnol 12(3):269–274

    Article  CAS  PubMed  Google Scholar 

  9. Sambrook J, Russell DW (2006) Calcium-phosphate-mediated transfection of eukaryotic cells with plasmid DNAs. CSH Protoc 2006(1)

    Google Scholar 

  10. Cleland WW (1964) Dithiothreitol, a new protective reagent for SH groups. Biochemistry 3:480–482

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ryan Jensen .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Jensen, R. (2014). Purification of Recombinant 2XMBP Tagged Human Proteins from Human Cells. In: Wajapeyee, N. (eds) Cancer Genomics and Proteomics. Methods in Molecular Biology, vol 1176. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0992-6_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-0992-6_17

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0991-9

  • Online ISBN: 978-1-4939-0992-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics