CoPurification of Interacting Proteins

These approaches are useful for identifying novel proteins that interact with a protein of interest, and for detecting time-specific interactions for proteins that have been shown by other methods to interact. Co-purification procedures are very similar to the purification protocols described in Subheading 3.3. However, co-purification should be performed using buffers with lower salt concentration than used in buffers in simple purification of the target protein. Relatively low salt concentration does not disrupt protein interactions and allows co-purification of the target protein and its interactants.

3.4.1. Affinity Co-Purification

The protocol is presented for His-tagged proteins, but other affinity tags and matrices can be substituted.

KaiC-His strain WT strain

Fig. 2. KaiA protein copurifies with His-tagged KaiC protein (see Subheading 3.4.1.). The proteins were eluted with 5 mL of IA elution buffer. Each 1-mL fraction was collected in a separate tube. Immunoblot of samples using antisera raised against KaiA protein is shown. Numbers correspond to the eluate fraction. NS, nonspecific band.

Fig. 2. KaiA protein copurifies with His-tagged KaiC protein (see Subheading 3.4.1.). The proteins were eluted with 5 mL of IA elution buffer. Each 1-mL fraction was collected in a separate tube. Immunoblot of samples using antisera raised against KaiA protein is shown. Numbers correspond to the eluate fraction. NS, nonspecific band.

1. Induce expression of the His-tagged protein if it is under control of a regulated promoter (see Note 6).

2. Prepare the cyanobacterial soluble protein extract as described in Subheadings 3.1.2. and 3.2., with the exception of substituting IA lysis buffer for PBS buffer.

3. Load the soluble protein fraction on a column prepacked with 2 mL Ni-NTA-agarose.

4. Wash the column five times with 10 mL of IA wash buffer.

5. Elute with 5 mL of IA elution buffer, collecting each 1-mL fraction in a separate tube (Fig. 2). Proceed with analysis of the eluted fractions to see which one has the highest concentration of target protein (see Note 7).

3.4.2. Coimmunoprecipitation

The following procedure was compiled from the conditions used by Kitayama et al. (8).

1. In advance of coimmunoprecipitation reactions, couple 25 ^L bed volume of AffiGel-Hz beads to purified antibodies raised to the protein of interest. To block nonspecific interactions wash the coupled resin with 0.5% BSA in IP buffer.

2. Harvest 0.1 g of cyanobacterial cells (about 100 mL of culture at optical density of 0.5 at 750 nm). Proceed with cell disruption and fractionation as described in Subheadings 3.1.2. and 3.2., with the exception of substituting TES-NaOH buffer for PBS.

3. Adjust the protein content of soluble extract for each sample to the same concentration (0.2 mg/mL is suggested). Proceed with 700 ^L of the soluble fraction.

4. Add 700 ^L of IP buffer. Incubate the resulting 1400 ^L with the coupled resin at 4°C for 2 h.

5. Wash the beads twice with 1 mL of IP buffer that contains 0.5% w/v BSA to minimize nonspecific interaction, and four times with 1 mL of IP buffer without BSA.

6. Resuspend the beads in 80 ^L of SDS-PAGE loading buffer without reducing agent. Elute proteins by vortexing gently for 10 min at room temperature. Spin briefly to collect the supernatant fraction.

7. Supplement the collected fraction with reducing agent (0.1% 2-mercaptoethanol) and 0.1% bromophenol blue to load on SDS-PAGE for further analysis (see Note 7).

4. Notes

1. The amount of cells used for this assay depends greatly on the sensitivity of the antibodies used. A series of 1:10 dilutions of concentrated culture may be used in the first experiment. Optical density measurements must be made at longer wavelengths for cyanobacteria than for Escherichia coli so that light scattering, rather than absorbance by the photosynthetic machinery, will be detected.

2. Addition of 1 mM phenylmethylsulfonyl fluoride (prepare stock solution of 100 mM in isopropanol) or 1/1000 vol of protease inhibitor cocktail for general use (Sigma) or other protease inhibitors (9) significantly improves stability of proteins in the extract. Phenylmethylsulfonyl fluoride permanently inhibits proteases, but it is quickly inactivated in aqueous solutions; therefore, it should be added directly to the sample containing proteases, not to the buffer before adding it to the sample. For long-term storage, freezing of the sample is recommended. Also, to decrease degradation rates of proteins during the protein extraction and purification protocols described in this chapter, it is recommended to use ice-cold buffers and keep the samples at 4°C.

3. A few methods for disrupting cyanobacterial cells have been described. Approaches other than breakage with glass beads as outlined in this chapter include sonication (3) or passing cells through a French pressure cell press (8).

4. If it is important to extract most of the total protein from the sample, and low protein concentration in the extract is not an issue (for example, when you are planning to proceed with protein purification; see Subheading 3.3.), add an additional 500 ^L of PBS after collecting the first supernatant fraction, spin briefly, and collect the supernatant fraction again. Repeat a few times until the supernatant fraction is clear. Proceed with centrifugation of the collected green supernatant fraction for 10 s at 1000g.

5. Methods for localization of proteins in the periplasm and thylakoid or plasma membranes of cyanobacteria are also available (12,13). Proteins from membrane fractions may also be separated by their hydrophilic or hydrophobic nature, and the strength of protein attachment to the membrane can be evaluated (14). Control proteins of known localization should be assayed to assure purity of the fractions. The protein fractionation methods mentioned here are based on solubility of proteins and their attachment to membranes. In addition, the cell extract can be subdivided based on size of proteins and protein complexes using gel filtration chromatography (3).

6. Affinity-tagged alleles are commonly driven in S. elongatus by a promoter under lac repressor control. Addition of 1 mM IPTG for 1 h is normally sufficient for protein expression controlled by pTrc promoter. However, concentration of IPTG and time of induction greatly depend on the individual protein, experimental variables, and the specific promoter, and should be adjusted accordingly. If the His-tagged protein is being expressed for purification of its possible interactants, it is important to keep expression levels relatively low, because gross overexpression may increase nonspecific protein interaction in the cell. Titration of IPTG with phenotypic monitoring should be used to keep the protein within physiologically relevant concentrations.

7. The eluted fractions can be analyzed by immunoblotting using the antibody raised against proteins known to be involved in circadian rhythmicity. The eluted fractions can be concentrated by precipitation of proteins with trichloroacetic acid. Add trichloroacetic acid to the sample to final concentration of 15%, freeze the sample at -80°C, then thaw and spin at 16,000g to precipitate the protein. Carefully remove all liquid and air-dry the sample. Resuspend the pellets in SDS gel-loading buffer and boil before loading on a SDS-PAGE gel for further staining with Coomassie brilliant blue (9). Co-purifying proteins may be identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

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