Protocol of Protein Purification

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Once the POI-3C-10His protein is extracted from cellular membranes in soluble form, it may be purified to obtain a sample that is Pure (free of other proteins and contaminants), Homogenous (single uniform population), Stable (typically over a week in concentrated form at 4 C), and Free of protein-free detergent micelles (this combined state will be referred to as ‘‘PHSF’’). To accomplish this, we employ a narrow range of techniques including immobilized metal affinity (IMAC), size-exclusion (SEC) and ion-exchange chromatography. These methods are synergistic, iterative, and employed to varying degrees depending on the target protein. For the current discussion, we will detail a standard approach of IMAC followed by cleavage of the expression tag, reverse-IMAC to remove uncleaved protein, and finally SEC to obtain the purified protein in diluted form. This sample will then be concentrated and analyzed prior to use. If this sample is intended for structure determination (i.e., crystallization) then special caution should be taken to avoid a significant concentration of protein-free detergent micelles (Newby et al., 2009). Thus, we will continue with the theme of purifying our target protein, POI-3C-10His, which was solubilized in the previous section. Recommended detergent concentrations for SEC buffers are as follows: 40 mM OG, 12 mM NG, 4 mM DM, 1 mM DDM, 12 mM LDAO, and 4 mM FC-12 (2 CMC is a good starting point for most detergents). For the current example, we will use 1 mM DDM in all buffers (as determined in Section 5). The initial step to protein purification is a metal-affinity purification of the solubilized membranes; we generally use 125 ml of Ni-NTA agarose resin (Qiagen) per mg of expected protein yield. The selected IMAC resin should be prepared according to manufacturer’s specifications and optimized as needed. The solubilized membranes should be incubated with IMAC resin at 4 C with nutation for at least 1 h though generally not longer than 3 h. We have found that the degree of target protein binding to Ni-NTA resin does not increase substantially past 3 h though increased proteolysis and binding of contaminant proteins may occur. Following incubation, the Ni-NTA resin containing bound POI-3C-10 His protein should be transferred to a gravity flow column and washed with 20 column volumes of Buffer A (20 mM Tris (pH 7.4, RT), 200 mM NaCl, 10% (v/v) glycerol, 4 mM b-ME, 1 mM PMSF, and 1 mM DDM) containing 10 mM Overexpression and Purification of Integral Membrane Proteins in Yeast 701 imidazole. If following the wash by absorbance, it is beneficial to wash until A280 nm returns to baseline. It is important at this point to obtain about 10 ml of initial flow-through for SDS–PAGE analysis. The above steps are repeated with Wash 2 and Wash 3 buffers. Finally, POI-3C-10 His is eluted from the column using the IMAC elution buffer. If possible, reduce the flow rate prior to elution to ensure the target protein elutes in a minimal volume. Be careful to observe the eluted sample for turbidity, especially over the ensuing several minutes as the protein may be unstable in the prescribed buffer and thus precipitate out of solution at this point. If precipitation occurs, one can make appropriate changes to the IMAC buffers (e.g., changing salt concentration or pH) to increase protein stability. It is also advisable to perform a buffer exchange immediately following elution into 20 mM HEPES (pH 7.4), 150 mM NaCl, 10% (v/v) glycerol, 4 mM b-ME, 1 mM PMSF, and 1 mM DDM (SEC buffer). This can be accomplished with a small desalting column such as the EconoPac 10 DG disposable chromatography column from Bio-Rad. Following IMAC and buffer exchange, the POI-3C-His protein is ready for cleavage of the linker-3C-10 His expression tag. There are a broad number of site-specific proteases for cleaving affinity tags, though care should be taken to ensure they are active in the prescribed detergent. The human rhinovirus 3C protease and thrombin are both robust and efficient proteases that have worked very well for cleaving affinity tags attached to detergent solubilized membrane proteins. We have had great success with an MBP-3C fusion construct described previously. To cleave the POI-3C-His affinity tag, the protein should be incubated overnight at 4 C with approximately a 1:5 ratio of protease to target protein in whatever volume of buffer is obtained in the desalting step above. Retain pre- and postcleavage 10 ml samples for SDS–PAGE gel analysis to evaluate cleavage. Following cleavage a reverse-IMAC purification (i.e., the flow-through is retained) is performed using metal-affinity resin to separate cleaved 3C-His tag and protease (which is also His tagged) from the target protein. This step entails a 1h incubation with IMAC resin, such as ‘‘Talon’’ metal-affinity resin, in batch at 4 C. Following incubation, the flow-through should be retained—this contains the cleaved POI protein that will be purified in the next step. Elute resin bound protein from the column using the IMAC elution buffer and collect a 10-ml sample for analysis on a gel to ascertain if nonspecific binding of the target protein is occurring. Following completion of this step, the Ni-purified, 3C-cleaved POI protein is now ready for further purification. Ion-exchange chromatography is a powerful technique that separates macromolecules based upon charge state at a given pH. Though not discussed within this chapter, we have often used this technique to purify difficult targets, concentrate protein, reduce protein-free detergent micelles, perform detergent exchanges, or obtain a pH stability profile. We generally use 1 or 5 ml disposable HiTrap sepharose Q or SP ion-exchange columns from GE Healthcare.