DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: A practical method for efficient and optimal production of Seleno‐methionine‐labeled recombinant protein complexes in the insect cells

Abstract

Abstract The use of Seleno‐methionine (SeMet) incorporated protein crystals for single or multi‐wavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X‐ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in Escherichia coli . In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet‐incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allow for the incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yieldmore » of 60–90% compared with native protein expression.« less

Authors:
 [1];  [1]; ORCiD logo [1]
  1. Department of Biochemistry and Molecular Biology Indiana University School of Medicine 635 Barnhill Drive, Indianapolis, Indiana 46202
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1493371
Resource Type:
Publisher's Accepted Manuscript
Journal Name:
Protein Science
Additional Journal Information:
Journal Name: Protein Science Journal Volume: 28 Journal Issue: 4; Journal ID: ISSN 0961-8368
Publisher:
Wiley Blackwell (John Wiley & Sons)
Country of Publication:
United Kingdom
Language:
English

Citation Formats

Wenzel, Sabine, Imasaki, Tsuyoshi, and Takagi, Yuichiro. A practical method for efficient and optimal production of Seleno‐methionine‐labeled recombinant protein complexes in the insect cells. United Kingdom: N. p., 2019. Web. doi:10.1002/pro.3575.
Wenzel, Sabine, Imasaki, Tsuyoshi, & Takagi, Yuichiro. A practical method for efficient and optimal production of Seleno‐methionine‐labeled recombinant protein complexes in the insect cells. United Kingdom. https://doi.org/10.1002/pro.3575
Wenzel, Sabine, Imasaki, Tsuyoshi, and Takagi, Yuichiro. Mon . "A practical method for efficient and optimal production of Seleno‐methionine‐labeled recombinant protein complexes in the insect cells". United Kingdom. https://doi.org/10.1002/pro.3575.
@article{osti_1493371,
title = {A practical method for efficient and optimal production of Seleno‐methionine‐labeled recombinant protein complexes in the insect cells},
author = {Wenzel, Sabine and Imasaki, Tsuyoshi and Takagi, Yuichiro},
abstractNote = {Abstract The use of Seleno‐methionine (SeMet) incorporated protein crystals for single or multi‐wavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X‐ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in Escherichia coli . In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet‐incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allow for the incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60–90% compared with native protein expression.},
doi = {10.1002/pro.3575},
journal = {Protein Science},
number = 4,
volume = 28,
place = {United Kingdom},
year = {2019},
month = {2}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1002/pro.3575

Citation Metrics:
Cited by: 3 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Baculovirus as versatile vectors for protein expression in insect and mammalian cells
journal, May 2005

  • Kost, Thomas A.; Condreay, J. Patrick; Jarvis, Donald L.
  • Nature Biotechnology, Vol. 23, Issue 5
  • DOI: 10.1038/nbt1095

Mediator as a General Transcription Factor
journal, November 2005

  • Takagi, Yuichiro; Kornberg, Roger D.
  • Journal of Biological Chemistry, Vol. 281, Issue 1
  • DOI: 10.1074/jbc.M508253200

Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing
journal, April 2007

  • Malkowski, M. G.; Quartley, E.; Friedman, A. E.
  • Proceedings of the National Academy of Sciences, Vol. 104, Issue 16
  • DOI: 10.1073/pnas.0610337104

Herpes Simplex Virus Glycoprotein D Bound to the Human Receptor HveA
journal, July 2001


Trans-sulfuration Pathway Seleno-amino Acids Are Mediators of Selenomethionine Toxicity in Saccharomyces cerevisiae *
journal, March 2015

  • Lazard, Myriam; Dauplais, Marc; Blanquet, Sylvain
  • Journal of Biological Chemistry, Vol. 290, Issue 17
  • DOI: 10.1074/jbc.M115.640375

Structural basis of eukaryotic gene transcription
journal, November 2004


A phased translation function
journal, October 1988


Mediator Head module structure and functional interactions
journal, February 2010

  • Cai, Gang; Imasaki, Tsuyoshi; Yamada, Kentaro
  • Nature Structural & Molecular Biology, Vol. 17, Issue 3
  • DOI: 10.1038/nsmb.1757

Advances in recombinant protein expression for use in pharmaceutical research
journal, June 2013

  • Assenberg, Rene; Wan, Paul T.; Geisse, Sabine
  • Current Opinion in Structural Biology, Vol. 23, Issue 3
  • DOI: 10.1016/j.sbi.2013.03.008

Architecture of the Mediator head module
journal, July 2011

  • Imasaki, Tsuyoshi; Calero, Guillermo; Cai, Gang
  • Nature, Vol. 475, Issue 7355
  • DOI: 10.1038/nature10162

Autographa californicaNucleopolyhedrovirus Infection Results in Sf9 Cell Cycle Arrest at G2/M Phase
journal, April 1998

  • Braunagel, Sharon C.; Parr, Rebecca; Belyavskyi, Michail
  • Virology, Vol. 244, Issue 1
  • DOI: 10.1006/viro.1998.9097

Crystal Structure of a Claudin Provides Insight into the Architecture of Tight Junctions
journal, April 2014


Highly efficient selenomethionine labeling of recombinant proteins produced in mammalian cells
journal, August 2006

  • Barton, William A.; Tzvetkova-Robev, Dorothea; Erdjument-Bromage, Hediye
  • Protein Science, Vol. 15, Issue 8
  • DOI: 10.1110/ps.062244206

Selenomethionyl analog of recombinant human choriogonadotropin
journal, May 1991


Cell-Cycle Perturbation in Sf9 Cells Infected withAutographa californicaNucleopolyhedrovirus
journal, May 1999


Crystal Structure of Mouse H2-M
journal, September 1998


Structure of the Reovirus Membrane-Penetration Protein, μ1, in a Complex with Its Protector Protein, σ3
journal, January 2002


Exposure to selenomethionine causes selenocysteine misincorporation and protein aggregation in Saccharomyces cerevisiae
journal, March 2017

  • Plateau, Pierre; Saveanu, Cosmin; Lestini, Roxane
  • Scientific Reports, Vol. 7, Issue 1
  • DOI: 10.1038/srep44761

Crystallographic analysis of CD40 recognition and signaling by human TRAF2
journal, July 1999

  • McWhirter, S. M.; Pullen, S. S.; Holton, J. M.
  • Proceedings of the National Academy of Sciences, Vol. 96, Issue 15
  • DOI: 10.1073/pnas.96.15.8408

Producing selenomethionine-labeled proteins with a baculovirus expression vector system
journal, November 1999


Genome-wide screen of Saccharomyces cerevisiae null allele strains identifies genes involved in selenomethionine resistance
journal, November 2008

  • Bockhorn, J.; Balar, B.; He, D.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 46
  • DOI: 10.1073/pnas.0805642105

Selenomethionine Incorporation in Saccharomyces cerevisiae RNA Polymerase II
journal, January 2001


Production of selenomethionyl-derivatized proteins in baculovirus-infected insect cells
journal, September 2007

  • Cronin, Ciarán N.; Lim, Kheng B.; Rogers, Joe
  • Protein Science, Vol. 16, Issue 9
  • DOI: 10.1110/ps.072931407

Mediator and the mechanism of transcriptional activation
journal, May 2005


CDK8: A positive regulator of transcription
journal, July 2010

  • Galbraith, Matthew D.; Donner, Aaron J.; Espinosa, Joaquín M.
  • Transcription, Vol. 1, Issue 1
  • DOI: 10.4161/trns.1.1.12373

Structure and TBP binding of the Mediator head subcomplex Med8–Med18–Med20
journal, September 2006

  • Larivière, Laurent; Geiger, Sebastian; Hoeppner, Sabine
  • Nature Structural & Molecular Biology, Vol. 13, Issue 10
  • DOI: 10.1038/nsmb1143

Analysis of Saccharomyces cerevisiae null allele strains identifies a larger role for DNA damage versus oxidative stress pathways in growth inhibition by selenium
journal, November 2008

  • Seitomer, Eden; Balar, Bharvi; He, Dongming
  • Molecular Nutrition & Food Research, Vol. 52, Issue 11
  • DOI: 10.1002/mnfr.200700347

G protein-coupled receptor overexpression with the baculovirus–insect cell system: a tool for structural and functional studies
journal, February 2003


[20] Processing of X-ray diffraction data collected in oscillation mode
book, January 1997


Protein complex expression by using multigene baculoviral vectors
journal, November 2006

  • Fitzgerald, Daniel J.; Berger, Philipp; Schaffitzel, Christiane
  • Nature Methods, Vol. 3, Issue 12
  • DOI: 10.1038/nmeth983

Phaser crystallographic software
journal, July 2007

  • McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
  • Journal of Applied Crystallography, Vol. 40, Issue 4
  • DOI: 10.1107/S0021889807021206

Use of novel selenomethionine-resistant yeast to produce selenomethionyl protein suitable for structural analysis
journal, May 2009