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Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels
 
Michal Domanski1, Kelly Molloy2, Hua Jiang3, Brian T. Chait2, Michael P. Rout3, Torben Heick Jensen1, and John LaCava3
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Supplementary Material
Protocol 1 (.pdf)
Protocol 2 (.pdf)

Results and discussion

Optimization of Dynabeads M270 antibody conjugate

Given the advantages of magnetic particles for affinity isolation, we generated our own anti-FLAG M2 (27, 43) antibody-coupled magnetic beads (MBs, Dynabeads M-270 epoxy; Invitrogen) and tested them via affinity isolation of the reporter protein BAP-FLAG (no. P7457; Sigma-Aldrich). The comparison to affinity isolations performed with commercially available agarose resins from Sigma-Aldrich (products A2220 and M8823) demonstrates several points of note (Figure 1 and Supplementary Figure S2). First, the MBs exhibit significantly reduced nonspecific protein binding when compared with agarose beads, even at low stringency of purification. Secondly, although Sigma-Aldrich offers two grades of anti-FLAG antibody, purified immunoglobulin (F3165) or affinity isolated antibody (F1804), we see no significant difference in performance between the two in our assay. Moreover, the F3165 product requires no preparative handling, whereas F1804 requires desalting to remove glycerol prior to coupling. While the F3165 product does contain NaN3 (0.02%), which can react with the epoxy functionalized M-270 Dynabeads used here, interference is minimal at the concentration used for antibody preservation (typically <10% reduction in performance; Figure 1, compare lanes 2 and 3). On the other hand, the F1804 product contains glycerol (50%), which significantly reacts with the epoxy-functionalized beads, necessitating desalting (data not shown). We observed a modest increase in the binding of our reporter protein, BAP-FLAG, with increasing concentration of the antibody used during conjugation over the range presented. We settled on conjugating at 10 μg/mg MBs directly, without desalting, using purified immunoglobulin (F3165) given the relative performance and antibody cost. The same strategy has been successfully applied to coupling polyclonal llama anti-GFP antibodies and anti-GFP nanobodies (see below), as well as anti-Myc E910 monoclonal antibodies (data not shown) to MBs.





Expression of tagged proteins at near endogenous levels

Due to the artifact prone nature of protein over-expression (1, 19-21) (Supplementary Figure S3), we set out to create cell lines expressing our proteins of interest at near endogenous levels via the HEK293 Flp-In T-REx system. We previously generated cell line derivatives stably expressing tetracycline-inducible versions of RRP6, RRP41, and SKIV2L2, each with a C-terminal FLAG-tag, respectively (17). For point of comparison in affinity isolation, this work was repeated using C′-terminal 3×FLAG-tag constructs. In all cases tested, the 3×FLAG-tagged proteins have exhibited superior affinity isolation in terms of total yield and the stringency at which the purification can be successfully carried out (Supplementary Figure S4). We have observed that 3×FLAG-tagged proteins can be readily affinity isolated in the presence of 1 M NaCl, 2 M urea, or 1% (w/v) N-laurylsarcosine, respectively, without apparent loss of yield for the handle protein (although many coprecipitating proteins are lost under these conditions; data not shown); hence we have adopted 3×FLAG-tags moving forward. Comparable results to those acquired with 3×FLAG have also been obtained using a 3×Myc-tag, in concert with the E910 monoclonal antibody (data not shown), but this lacks a well-established method for native elution by competitive displacement achievable with the 3×FLAG-tag (26). As can be seen in Figure 2, RRP6-3×FLAG was readily titrated to near the endogenous level by using tetracycline at a concentration of 5 ng/mL in the media, demonstrating the ability of this system to achieve physiological expression of proteins of interest. An alternative approach is to express your protein of interest as a stable, affinity-tagged, BAC-cloned transgene from the endogenous promoter and context (18).





Cryogenic disruption of human cells shows favorable properties for protein affinity isolation using magnetic beads

In order to characterize the relative merits of cell disruption by cryogenic grinding versus standard sonication in human cells, we utilized anti-GFP conjugated MBs and HeLa Kyoto cells, expressing the EGFP-containing RBM7-LAP protein (13, 14) (Figure 3). Each experiment utilized equivalent material from only one ~90% confluent 150-cm2 culture dish, which is ~100 mg WCW in the case of cryogenic cell grindate. The control cell line clearly shows that sonication generates a vastly increased level of nonspecific protein binding in the absence of the expressed tagged protein. In stark contrast, although some nonspecific binding remains apparent in the sonicated sample when RBM7-LAP is expressed, the differences between sonicated and cryogenically disrupted cells are much less pronounced. Nonspecific binding of protein to affinity resins can vary significantly with extraction and washing solution compositions, but these data suggest that mechanical grinding provides increased uniformity between the tagged protein expressing and control cell lines. Given these and the above observations, we conclude that cryogenic disruption of cells followed by affinity isolation using magnetic beads is highly efficient, reliable, and robust. Figure 4 shows a panel of purifications using tagged proteins purified via the methods optimized as described here. The exosome and NEXT complexes were eluted with LDS sample loading buffer at 75°C, while the CBC was eluted with 3×FLAG peptide to avoid contamination of the sample with IgG, which can occur when eluting with sample buffer at elevated temperature.

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