| Title |
Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5
|
|---|---|
| Published in |
Molecular Biotechnology, September 2018
|
| DOI | 10.1007/s12033-018-0107-2 |
| Pubmed ID | |
| Authors |
Monika M. Golas, Sakthidasan Jayaprakash, Le T. M. Le, Zongpei Zhao, Violeta Heras Huertas, Ida S. Jensen, Juan Yuan, Bjoern Sander |
| 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. |
X Demographics
The data shown below were collected from the profiles of 5 X users who shared this research output. Click here to find out more about how the information was compiled.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| United Kingdom | 1 | 20% |
| Unknown | 4 | 80% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Members of the public | 4 | 80% |
| Scientists | 1 | 20% |
Mendeley readers
The data shown below were compiled from readership statistics for 13 Mendeley readers of this research output. Click here to see the associated Mendeley record.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 13 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Ph. D. Student | 3 | 23% |
| Researcher | 3 | 23% |
| Student > Master | 2 | 15% |
| Professor | 1 | 8% |
| Other | 1 | 8% |
| Other | 0 | 0% |
| Unknown | 3 | 23% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 4 | 31% |
| Pharmacology, Toxicology and Pharmaceutical Science | 1 | 8% |
| Agricultural and Biological Sciences | 1 | 8% |
| Immunology and Microbiology | 1 | 8% |
| Sports and Recreations | 1 | 8% |
| Other | 1 | 8% |
| Unknown | 4 | 31% |
Attention Score in Context
This research output has an Altmetric Attention Score of 3. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 14 November 2018.
All research outputs
#13,901,936
of 23,577,654 outputs
Outputs from Molecular Biotechnology
#600
of 999 outputs
Outputs of similar age
#173,030
of 336,822 outputs
Outputs of similar age from Molecular Biotechnology
#2
of 17 outputs
Altmetric has tracked 23,577,654 research outputs across all sources so far. This one is in the 39th percentile – i.e., 39% of other outputs scored the same or lower than it.
So far Altmetric has tracked 999 research outputs from this source. They receive a mean Attention Score of 3.6. This one is in the 39th percentile – i.e., 39% of its peers scored the same or lower than it.
Older research outputs will score higher simply because they've had more time to accumulate mentions. To account for age we can compare this Altmetric Attention Score to the 336,822 tracked outputs that were published within six weeks on either side of this one in any source. This one is in the 46th percentile – i.e., 46% of its contemporaries scored the same or lower than it.
We're also able to compare this research output to 17 others from the same source and published within six weeks on either side of this one. This one has done well, scoring higher than 88% of its contemporaries.