Finally, as with Bio-Ox (8), PEGylOx may be depleted from samples by spin-column-based gel filtration chromatography (Supplementary Protocol). We assayed the level of depletion for PEGylOx and found greater than 100× depletion of the peptide through the application of eluted samples to desalting / buffer exchange spin columns with a 40 kDa molecular weight cut-off (MWCO) as assayed by UV280 and Coomassie staining. We tested Bio-Rad (Hercules, CA) Bio-Gel P-30 (#732–6223) and Thermo (Waltham, MA) Zeba 40K MWCO (#87764) spin columns and achieved equivalent results (Supplemetary Section S5). Resins with lower MWCOs that we tested (Bio-Rad Bio-Gel P-6 #732–6221 and Thermo Zeba 7K MWCO #89877; UV280 data for P-6 in Supplementary Section S5) were only able to deplete PEGylOx by ∼10–20×. In contrast, we were unable to significantly deplete 3xFLAG peptide in this manner (data not shown). Hence PEGylOx also exhibits the beneficial property of being depleted from samples by desalting / buffer exchange when needed for downstream applications.
Unfortunately, the optimization of affinity isolation / native elution often requires attention to a very broad range of parameters – pH, overall ionic strength, salt-type(s) and concentration, detergent-type(s) and concentration, time, and temperature, among others. Optimized parameters for the capture of a given complex from cell extract will often need to be determined empirically through trial and error – but some prior knowledge about the requirements of the affinity system can help provide boundaries for exploring new isolation conditions.
Protein affinity isolation is typically carried out at 4°C, which retards the disintegration of most protein complexes and helps reduce enzyme activity within the cell extract; this includes proteolysis – although an appropriate cocktail of protease inhibitors should also be included. Solvents for extracting protein complexes from cells typically employ a near physiological pH (∼7.0–8.0 based on the pH of the cytosol), although there is good reason to vary this parameter for complexes believed to reside in cellular compartments of differing physiological pH. In this study, we have constrained our pH to a range we know works well for the SpA affinity tag. The canonical SpA interaction with Human IgG Fc occurs through an induced hydrophobic fit, which is weakened as the pH is shifted to acidic values. This pH sensitivity is exploited to fractionate differing IgG subclasses from one another on SpA affinity media (16-19). Hence, a slightly alkaline pH promotes the capture and retention of SpA-tagged proteins on IgG media while still remaining within the typical physiological range. This phenomenon bears a connection to the number of SpA domains present (18), and we have observed that the affinity of tandem repeats of the Z-domain for IgG is significantly diminished at pH ∼6.0 and below (20). Moreover, since PEGylOx interacts with Fc through a highly conserved mechanism (11), its optimal binding conditions are likely to be very similar to those mentioned above. Along these lines, we have successfully achieved competitive displacement of protein complexes using PEGylOx in HEPES buffered solution at pH 7.4 and ammonium acetate, pH 7.0 – with results comparable to those presented here using Tris pH 8.0 (data not shown). Finally, keep in mind that the multiplicity and variety of IgG binding domains present in different SpA-tag configurations may factor into performance through avidity effects (2, 18, 21, 22) (Supplementary Section S4B).
Through affinity isolation and subsequent washing, the complex of interest is purified away from the bulk cell extract and thus away from the majority of proteolytic activity. At this point, the importance of low temperature in maintaining the integrity of the protein complex may be somewhat diminished. For many of our complexes we carry out brief post-capture washes and a subsequent 15 min incubation with PEGylOx at RT (Supplementary Protocol). This is primarily a matter of convenience, although elution is expected to occur more rapidly as temperature increases (i.e., at RT versus 4°C). The precise extent of the difference in elution efficiency can vary from complex to complex (8), but going above RT could have the undesirable side effect of accelerating the disintegration of the complex of interest. In cases where we observe that complex components are labile when working at RT, we make every effort to work at between 0°-4°C. However, in such cases, we take this as an indicator that further optimization of the extraction solvent may provide greater stability during RT handling. For this work, the following extraction and washing solutions were used: Nup53p and Nup1p – 40 mM Tris-Cl buffer solution at pH 8.0 with 50 mM trisodium citrate, 300 mM NaCl, 0.1% v/v Tween 20, and 1 mM EDTA (pH 8.0); Psf2p – 20 mM HEPES-Na buffered solution at pH 7.4 with 150 mM NaCl, 2 mM MgCl2, 0.1% v/v Tween 20; RpoC – HEPES-Na buffered solution at pH 7.4 with 150 mM NaCl, 2 mM MgCl2, 0.1 mM CaCl2, and 0.1% v/v Tween 20, including the addition of DNase I to extracts.
This work was funded by the National Institutes of Health (NIH) / National Center for Research Resources (NCRR) grant no. U54 GM103511. We thank Zachary Quinkert for stimulating discussion; Lars Westblade, Seth Darst, and Robert Landick for providing the RpoC-SpA E. coli strain and useful information; and Brian Chait for providing the Psf2p-SpA strain, and for stimulating discussions.
The authors declare no competing interests.
Address correspondence to Michael P. Rout, Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, USA. E-mail: [email protected]
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