首页 » 实验方法 » 蛋白实验 » 蛋白纯化

Purification of Human Multiprotein Complexes using OneSTrEP Technology

来源:生物谷  2010/2/5   访问量:8727
评论(0)
分享

Introduction

Here we describe a strategy for isolation of multiprotein complexes from human HeLa S3 cells in a scale and purity optimized for characterization by mass spectrometry. For this purpose, we use stably expressed One-STrEP-tag ® fusion proteins. This approach was successfully used in characterization of histone chaperone Asf1 complexes (Groth et al., 2007), and we have recently optimized it further. Using this protocol we routinely obtain complexes in amounts sufficient for visualising single protein bands by Coomassie Blue staining (Figure 1).

The Strep-tag®II (SAWRHPQFGG) and its “double” sister, the One-STrEP-tag (tandem arrangement of Strep-tag®II, here called OneStrep), are reasonably small protein tags that usually do not influence protein folding and function. However, always check by appropriate control experiments that the tagged protein is functional in vivo. Vectors for both N- and C-terminal fusion are available from IBA Tagnology (Germany), who has developed these tags and binding matrices. We recommend using the OneStrep-tag for purification of protein complexes from mammalian cells. The Strep-tag® has a strong affinity towards engineered streptavidin (Strep-Tactin®) which allows efficient one-step purification with high stringency washing to obtain highly pure protein complexes. Elution of the complexes by competition with D-biotin is efficient and can be done in a variety of buffer conditions allowing biochemical active complexes to be preserved. For more theoretical background and a protocol for purification of recombinant Strep-tag® fusion proteins from E.coli please refer to Schmidt and Skerra (2007).

In comparison to other commonly used purification strategies (i.e. HA and FLAG double-tag purification, Tagami et al., 2004), the advantages of the OneStrep-tag are:

  1. Purification in only one step;
  2. Strong affinity to Strep-Tactin® matrix allowing stringent washing;
  3. Highly efficient elution;
  4. Broad range of available Strep-Tactin® matrices including columns for gravity flow and HPLC as well as magnetic beads and spin-columns for small-scale purification;
  5. And cost-effectiveness.
Note that a two-step purification strategy may be advantageous to identify weak interactors.

Back to top

Procedure

Generation of HeLa S3 expressing YPI (Your Protein of Interest) tagged with OneStrep.

We strongly recommend that you generate stable cell lines rather than working with transiently transfected cells. This will reduce the non-physiological interaction due to overexpression and allow time for YPI-OneStrep to be incorporated into even highly stable complexes. After transfection of HeLa S3 cells with plasmids carrying the OneStrep fused to N- or C-terminal of YPI, clones can be isolated in 96 wells following 2 weeks of G418 selection (note 1). We recommend to isolate several clones and characterize them by a few simple criteria:

  1. Western blotting to check expression level (see protocol) and proper cellular distribution of OneStrep-YPI (note 2). Antibodies against YPI should allow to determine the extent of overexpression as the tag introduces a mobility shift;
  2. FACS analysis of DNA content to check the cell cycle profile;
  3. Viability and cell growth (i.e. using Trypan blue and cell counting).

Protein extracts

We use a protocol for making cytosolic and nuclear extracts from HeLa S3 cells grown on dishes that has been developed in the Almouzni laboratory (modified from Li and Kelly (1984) and Martini et al. (1998)). These extracts are competent for in vitro DNA replication (Li and Kelly, 1984) and nucleosome assembly assays (Martini et al. 1998; Groth et al., 2005). In principal, any of your favourite lysis buffer could be used, but it is important to check that none of the components interfere with binding of the OneStrep-tag to the Strep-Tactin® matrix (IBA Tagnology website: http://www.iba-go.com/prottools/prot_fr01_01.html). We recommend the pre-fractionation step as it reduces the complexity of the starting material and can provide functional insights if the cytosolic and nuclear extract yield distinct complexes.

  1. Grow cells on 150mm dishes until 80-90% confluence. We routinely use 6-8 150mm plates/experiment (note 3 and note 4).
  2. All following steps are done at 4°C in a cold room. Remove the medium and rinse cells 2x with ice-cold PBS. Drain well after the second wash.
  3. Add 10 ml Buffer E (-inhib.)/150mm plate and incubate for 8-10 min to swell the cells.
  4. Remove, drain and add 10ml of Buffer E (+inhib.)/150mm plate. Incubate for 8-10 min.
  5. Remove and drain well by leaving the dishes in vertical position for about 2 min.
  6. Scrape the cells and transfer into douncer (1 ml, Wheaton).
  7. Homogenize using a loose pestle with 25 strokes.
  8. Transfer to 2 ml Eppendorf tubes and pellet the nuclei by centrifugation at 1500 g for 5 min.
  9. Transfer the supernatant to a new tube and clear it by spinning 15 min in a tabletop centrifuge at 4°C max. speed (13.000 rpm). This is your cytosolic extract containing cytoplasmatic and soluble nuclear proteins. Snap freeze and store at -80°C.
  10. To the nuclei pellet from step 8 add one to two volumes of Buffer N and resuspend carefully with a P1000 pipette. Extract nuclear and chromatin bound proteins by end-over-end rotation 90 min at 4°C.
  11. Spin 15 min in a tabletop centrifuge at 4°C max. speed. Transfer the supernatant to a new tube and snap freeze. This is your nuclear extract containing nuclear and chromatin bound proteins. (note 5)
  12. Wash the pellet (matrix and salt-extracted chromatin) once with Buffer N, spin again, and snap freeze.

OneStrep-tag purification

We routinely use gravity flow Strep-Tactin Superflow® columns with a column volume (CV) of 200 µl (IBA Tagnology, Cat. No. 2-1207-550) (see note 6). Purification is performed at 4°C in a cold room.

  1. Equilibrate the column by applying 2x CV of Washing buffer.
  2. Add 5-10 mg of protein extracts to the column (not more than 1 ml). note 7
  3. Wash columns 10 times with 1 ml Washing buffer.
  4. Elute 6 times with 0,5 CV (100 µl)/elution step. note 8 and note 9
  5. Snap freeze eluted fractions E1-E6. For the first test purification, collect also the flow-through and washes for the analyses described below.

[1] [2] [3]  下一页

相关蛋白纯化