Streptavidin-Binding Peptide (SBP)-tagged SMC2 allows single-step affinity fluorescence, blotting or purification of the condensin complex

Abstract

BackgroundCell biologists face the need to rapidly analyse their proteins of interest in order to gain insight into their function. Often protein purification, cellular localisation and Western blot analyses can be multi-step processes, where protein is lost, activity is destroyed or effective antibodies have not yet been generated.AimTo develop a method that simplifies the critical protein analytical steps of the laboratory researcher, leading to easy, efficient and rapid protein purification, cellular localisation and quantification.ResultsWe have tagged the SMC2 subunit of the condensin complex with the Streptavidin-Binding Peptide (SBP), optimising and demonstrating the efficacious use of this tag for performing these protein analytical steps. Based on silver staining, and Western analysis, SBP delivered an outstanding specificity and purity of the condensin complex. We also developed a rapid and highly specific procedure to localise SBP-tagged proteins in cells in a single step procedure thus bypassing the need for using antibodies. Furthermore we have shown that the SBP tag can be used for isolating tagged proteins from chemically cross-linked cell populations for capturing DNA-protein interactions.ConclusionsThe small 38-amino acid synthetic SBP offers the potential to successfully perform all four critical analytical procedures as a single step and should have a general utility for the study of many proteins and protein complexes.

Ribosomal RNA Gene Transcription Mediated by the Master Genome Regulator Protein CCCTC-binding Factor (CTCF) Is Negatively Regulated by the Condensin Complex

“…In a previous report, affinity purification of SMC2 protein complex by streptavidin-binding peptide-tagged SMC2 could not detect CTCF (43). In our study, because of technical improvements, we performed FLAG and HA double-affinity protein purification for FLAG-HA-CTCF expressed in HeLa-S3 cells, cut the whole lane of PAGE gel, and performed MS.…”

Disruption of a Conserved CAP-D3 Threonine Alters Condensin Loading on Mitotic Chromosomes Leading to Chromosome Hypercondensation

“…Thr-1403 was mutated to Ala-1403 in CAP-D3 cDNA, orthologous to the Cdk1 site in human CAP-D3 at Thr-1415 (29). A streptavidin-binding peptide (SBP) GFP tag was engineered into the chicken CAP-D3 DNA at the C terminus for localization and purification purposes (33). The CAP-D3-T1403A SBP-GFP construct was then transfected into CAP-D3 KO cells.…”

“…Pulldown assay was performed as described before using asynchronous cells (29,33). Antibodies used were the rabbit anti-CAP-D3 (1:2500), mouse anti-phosphothreonine-proline (1:5000, CST), mouse anti-␣-tubulin (1:1000, Sigma), with goat anti-rabbit IgG-HRP and rabbit anti-mouse IgG-HRP as secondary antibodies (1:5000).…”

Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

“…Because of the high affinity and specificity of the SBP tag to streptavidin, this system has been successfully applied for one-step affinity purification of proteins [5], [7], [26], for studying protein-protein interactions [26], [27] and for developing improved tandem tags in protein studies [6], [27], [28], [29]. To make this powerful technology to be cost effective, development of an idealized streptavidin mutein, SAVSBPM18, is essential.…”