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 »
- Authors:
-
[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 = {Mon Feb 04 00:00:00 EST 2019},
month = {Mon Feb 04 00:00:00 EST 2019}
}
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1002/pro.3575
https://doi.org/10.1002/pro.3575
Other availability
Search WorldCat to find libraries that may hold this journal
Cited by: 1 work
Citation information provided by
Web of Science
Web of Science
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
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
Mediator as a General Transcription Factor
journal, November 2005
- Takagi, Yuichiro; Kornberg, Roger D.
- Journal of Biological Chemistry, Vol. 281, Issue 1
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
Herpes Simplex Virus Glycoprotein D Bound to the Human Receptor HveA
journal, July 2001
- Carfı́, Andrea; Willis, Sharon H.; Whitbeck, J. Charles
- Molecular Cell, Vol. 8, Issue 1
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
Structural basis of eukaryotic gene transcription
journal, November 2004
- Boeger, Hinrich; Bushnell, David A.; Davis, Ralph
- FEBS Letters, Vol. 579, Issue 4
A phased translation function
journal, October 1988
- Read, R. J.; Schierbeek, A. J.
- Journal of Applied Crystallography, Vol. 21, Issue 5
Mediator Head module structure and functional interactions
journal, February 2010
- Cai, Gang; Imasaki, Tsuyoshi; Yamada, Kentaro
- Nature Structural & Molecular Biology, Vol. 17, Issue 3
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
Architecture of the Mediator head module
journal, July 2011
- Imasaki, Tsuyoshi; Calero, Guillermo; Cai, Gang
- Nature, Vol. 475, Issue 7355
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
Crystal Structure of a Claudin Provides Insight into the Architecture of Tight Junctions
journal, April 2014
- Suzuki, H.; Nishizawa, T.; Tani, K.
- Science, Vol. 344, Issue 6181
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
Selenomethionyl analog of recombinant human choriogonadotropin
journal, May 1991
- Chen, W. Y.; Bahl, O. P.
- Journal of Biological Chemistry, Vol. 266, Issue 15
Cell-Cycle Perturbation in Sf9 Cells Infected withAutographa californicaNucleopolyhedrovirus
journal, May 1999
- Ikeda, Motoko; Kobayashi, Michihiro
- Virology, Vol. 258, Issue 1
Crystal Structure of Mouse H2-M
journal, September 1998
- Fremont, Daved H.; Crawford, Frances; Marrack, Philippa
- Immunity, Vol. 9, Issue 3
Structure of the Reovirus Membrane-Penetration Protein, μ1, in a Complex with Its Protector Protein, σ3
journal, January 2002
- Liemann, Susanne; Chandran, Kartik; Baker, Timothy S.
- Cell, Vol. 108, Issue 2
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
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
Producing selenomethionine-labeled proteins with a baculovirus expression vector system
journal, November 1999
- Bellizzi, John J.; Widom, Joanne; Kemp, Christopher W.
- Structure, Vol. 7, Issue 11
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
Selenomethionine Incorporation in Saccharomyces cerevisiae RNA Polymerase II
journal, January 2001
- Bushnell, David A.; Cramer, Patrick; Kornberg, Roger D.
- Structure, Vol. 9, Issue 1
The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus
journal, June 2009
- Wasilko, David J.; Edward Lee, S.; Stutzman-Engwall, Kim J.
- Protein Expression and Purification, Vol. 65, Issue 2
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
Mediator and the mechanism of transcriptional activation
journal, May 2005
- Kornberg, Roger D.
- Trends in Biochemical Sciences, Vol. 30, Issue 5
CDK8: A positive regulator of transcription
journal, July 2010
- Galbraith, Matthew D.; Donner, Aaron J.; Espinosa, Joaquín M.
- Transcription, Vol. 1, Issue 1
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
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
Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure.
journal, May 1990
- Hendrickson, W. A.; Horton, J. R.; LeMaster, D. M.
- The EMBO Journal, Vol. 9, Issue 5
G protein-coupled receptor overexpression with the baculovirus–insect cell system: a tool for structural and functional studies
journal, February 2003
- Massotte, Dominique
- Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1610, Issue 1
[20] Processing of X-ray diffraction data collected in oscillation mode
book, January 1997
- Otwinowski, Zbyszek; Minor, Wladek
- Macromolecular Crystallography Part A, 307-326
Protein complex expression by using multigene baculoviral vectors
journal, November 2006
- Fitzgerald, Daniel J.; Berger, Philipp; Schaffitzel, Christiane
- Nature Methods, Vol. 3, Issue 12
Phaser crystallographic software
journal, July 2007
- McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
- Journal of Applied Crystallography, Vol. 40, Issue 4
Use of novel selenomethionine-resistant yeast to produce selenomethionyl protein suitable for structural analysis
journal, May 2009
- Kitajima, Toshihiko; Yagi, Emi; Kubota, Tomomi
- FEMS Yeast Research, Vol. 9, Issue 3
Titer estimation for quality control (TEQC) method: A practical approach for optimal production of protein complexes using the baculovirus expression vector system
journal, April 2018
- Imasaki, Tsuyoshi; Wenzel, Sabine; Yamada, Kentaro
- PLOS ONE, Vol. 13, Issue 4