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Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5

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Abstract

The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins. Here, we demonstrate that human full-length SNF5 tagged with a C-terminal 3 × FLAG can be expressed and purified from insect cell extracts in monomeric and dimeric forms. To this end, we constructed a set of donor and acceptor vectors for the expression of individual proteins and protein complexes in the baculovirus/insect cell expression system under the control of a polyhedrin (polh), p10, or a minimal Drosophila melanogaster Hsp70 promoter. We show that the SNF5 expression level could be modulated by the selection of the promoter used to control expression. The vector set also comprises vectors that encode a 3 × FLAG tag, Twin-Strep tag, or CBP-3 × FLAG-TEV-ProteinA triple tag to facilitate affinity selection and detection. By gel filtration and split-ubiquitin assays, we show that human full-length SNF5 has the ability to self-interact. Overall, the toolbox developed herein offers the possibility to flexibly select the promoter strength as well as the affinity tag and is suggested to advance the recombinant expression of chromatin remodeling factors and other challenging proteins.

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Availability of the Toolbox

The toolbox will be deposited at the non-profit plasmid repository Addgene, and distributed to the scientific community.

Abbreviations

AcNPV:

Autographa californica nuclear polyhedrosis virus

ATP:

Adenosine triphosphate

BAF:

BRM/BRG1-associated factor

BAF47:

BRM/BRG1-associated factor 47

BRG1:

Brahma-related gene 1

BRM:

Brahma Abbreviations

CBP:

Calmodulin-binding peptide

CC:

Coiled coil

CE:

Cytoplasmic extract

DBD:

DNA-binding domain

DTT:

Dithiothreitol

E:

Elution fraction

EDTA:

Ethylenediaminetetraacetic acid

FBS:

Fetal bovine serum

FT:

Flow through

GST:

Glutathione S-transferase

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

HSE:

Heat shock element

Hsp:

Heat shock protein

INI1:

Integrase interactor 1

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

kDa:

Kilo dalton

LB:

Lysogeny broth

M:

Marker

MBP:

Maltose-binding protein

MCS:

Multiple cloning site

MDa:

Mega dalton

NE:

Nuclear extract

NMDA:

N-methyl-d-aspartate

OD600 :

Optical density at 600 nm

ORF:

Open reading frame

PAGE:

Polyacrylamide gel electrophoresis

PCR:

Polymerase chain reaction

Polh :

Polyhedrin

P/S:

Penicillin/streptomycin

SD:

Synthetic defined

SDS:

Sodium dodecyl sulfate

SMARCB1:

SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1

SNF5:

Sucrose Non Fermentable 5

SWI/SNF:

Switch/sucrose non-fermentable

TATA:

TATA box

TCA:

Trichloroacetic acid

TEV:

Tobacco etch virus

W:

Wash fraction

X-gal:

5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside

YFP:

Yellow fluorescent protein

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Acknowledgements

We wish to thank Susanne Stubbe and Golshah Ayoubi for expert technical assistance, and Srdja Drakulic for support with some of the experiments. We are grateful for the access to experimental facilities at the Danish Neuroscience Centre House, Aarhus University, Denmark. This study was supported by the Sapere Aude Program of the Danish Council for Independent Research, the Lundbeck Foundation’s Fellowship Program, the A.P. Møller Foundation for the Advancement of Medical Sciences, and the Carlsbergfondet to MMG. ISJ was supported by a fellowship of the Graduate School of Health, Aarhus University.

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Author notes

  1. Bjoern Sander

    Present address: Institute of Pathology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany

Authors and Affiliations

  1. Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 3, Building 1233, 8000, Aarhus C, Denmark

    Monika M. Golas, Sakthidasan Jayaprakash, Zongpei Zhao, Violeta Heras Huertas, Ida S. Jensen & Juan Yuan

  2. Stereology and Electron Microscopy Laboratory, Aarhus University, Wilhelm Meyers Allé 3, Building 1233, 8000, Aarhus C, Denmark

    Le T. M. Le & Bjoern Sander

  3. Center for Stochastic Geometry and Advanced Bioimaging, Aarhus University, Wilhelm Meyers Allé 3, Building 1233, 8000, Aarhus C, Denmark

    Bjoern Sander

  4. Department of Human Genetics, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany

    Monika M. Golas

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  1. Monika M. Golas
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Golas, M.M., Jayaprakash, S., Le, L.T.M. et al. Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5. Mol Biotechnol 60, 820–832 (2018). https://doi.org/10.1007/s12033-018-0107-2

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