Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5.
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.
['Golas|Monika M|MM|', 'Jayaprakash|Sakthidasan|S|', 'Le|Le T M|LTM|', 'Zhao|Zongpei|Z|', 'Heras Huertas|Violeta|V|', 'Jensen|Ida S|IS|', 'Yuan|Juan|J|', 'Sander|Bjoern|B|']
D000818:Animals D016367:Baculoviridae D002460:Cell Line D029721:Drosophila Proteins D004331:Drosophila melanogaster D015870:Gene Expression D018840:HSP70 Heat-Shock Proteins D006801:Humans D011401:Promoter Regions, Genetic D015202:Protein Engineering D000071796:SMARCB1 Protein D061987:Sf9 Cells D020798:Two-Hybrid System Techniques
Chromatin remodeler Recombinant protein Protein production Nucleus Plasmid Protein–protein interaction