2Laboratory Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY, USA
3Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY, USA
Here we present a streamlined affinity isolation approach for the analysis of human protein complexes. A synthesis of methods is used to achieve quality results without the protein over-expression typically required by such experiments. We have chosen several protein complexes related to RNA biology as examples to present the method, and we focus on the use of the triple-FLAG (3×FLAG) and GFP-tags and cryogenic grinding with cell lines that express the protein of interest at near endogenous levels. This method provides for high yield, low background affinity isolations using only modest quantities of cell material.
An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous-level tagged proteins. Isolations of triple-FLAG and GFP-tagged fusion proteins involved in RNA metabolism are presented.
Affinity isolation of proteins and associated complexes has facilitated the rapid growth of proteomic analyses (1). Model organisms amenable to targeted genetic engineering, such as the yeast Saccharomyces cerevisiae, have been at the center of this development (2-7)—providing a simple, direct path for the capture of endogenous protein complexes, ideal for both proteomic and biochemical analyses (8). While gene targeting systems for mammalian cell-types have been around for decades (9-12), simple, efficient methods for expressing affinity-tagged fusion proteins at or near endogenous levels are a more recent development (13-18). Moreover, this development was necessary; it is widely acknowledged that protein over-expression can lead to experimental artifacts, including mislocalization, and the formation of spurious interactions or altered activities (1, 19-21). Thus, for biomedical studies it is critical that tagged proteins be expressed at physiological levels.
Simultaneously, the technologies and methods for cell breakage and affinity isolation have themselves evolved. Our laboratory has shown that near complete cell breakage can be achieved by mechanical grinding at liquid N2 temperatures, resulting in a fine, granular material (cell grindate) with excellent properties for subsequent affinity isolation of tagged proteins and complexes (22-24) (e.g., http://lab.rockefeller.edu/rout/media/grinding.html). With respect to affinity isolations, the triple-FLAG (3×FLAG)-tag exhibits superiority to the single FLAG-tag through increased avidity of interaction with M2 anti-FLAG antibodies (25-27). We have observed that in many cases 3×FLAG-tagged protein complexes can be isolated at stringencies where traditional FLAG-tagged proteins fail to be immunoprecipitated. In addition, GFP makes an excellent tag not only for protein localization studies but also for the reliable capture of protein complexes (13, 22) since high-quality anti-GFP antibodies are available. Here we present our own polyclonal anti-GFP immunoglobulin as well as a bacterially expressed anti-GFP nanobody (28, 29). Further, antibody coupled, micron-scale magnetic beads have been indispensable in the development of high-yield, high-fidelity isolations of protein complexes from a variety of cell-types and using various affinity-tags (22-24).
We have applied these tools in a streamlined approach for the optimization of expression and capture of proteins involved in human RNA metabolism including the exosome complex, the NEXT complex, and the nuclear cap binding complex (CBC). The eukaryotic exosome complex has been shown to play a central and evolutionarily conserved role in the processing and degradation of a broad array of RNA species. Commensurate with its diverse activities, the exosome varies in composition along with cellular localization and requires activating cofactors (30). The NEXT complex, composed of RBM7, ZCCHC8, and SKIV2L2, also known as hMTR4, has recently been shown to target the exosome for the specific degradation of promoter upstream transcripts (PROMPTs) (17). However, the exosome and its cofactors have proven difficult to isolate from human cells at high yield and purity using modest quantities of starting material. Published methods have typically required long incubations and handling times during affinity isolation, and provided only silver stainable quantities of complex, requiring liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses or significant scale-up (16, 17, 31). Using a robust and rapid procedure of less than 2 h from extraction to elution that typically requires only 100 mg or less of starting material (wet cell weight; WCW), we affinity isolate the multicomponent human RNA exosome complex via both 3×FLAG-tagged RRP6 and RRP41 subunits, respectively, as well as the exosome cofactor NEXT complex, via LAP-tagged RBM7. These purifications provide Coomassie-stainable yield of coprecipitating proteins at high purity and apparent stoichiometry and are amenable to standard peptide mass fingerprinting analyses by MALDI-TOF MS. Finally, via a 3×FLAG-tagged NCBP2 (CBP20), we extend this strategy to the human CBC, important for pre-mRNA splicing and nuclear export of 5′-7-methyl guanosine (m7G) capped RNAs (32-35). In addition to the expected coprecipitation of NCBP1 (CBP80), we identify the SRRT, KPNA2, and PHAX proteins. This general strategy may provide for more facile analyses of human protein-protein interaction networks.