Recombinant CpcE and CpcF

1. The cpcE and cpcF genes overexpressed using a T7/pET vector system and the majority of the recombinant proteins are found in inclusion bodies.

2. The inclusion bodies are collected by low-speed centrifugation after the cells have been lysed by passage through a French pressure cell. The inclusion bodies appear as a chalky white pellet and are easily differentiated from unbroken cells which usually appear more tan or brownish in color.

3. The inclusion bodies should be washed extensively using the following solutions: 50 mM Tris-HCl, 5 mM EDTA, pH 8.0; 50 mM Tris-HCl, pH 8.0, 1% Triton® X-100; 50 mM Tris-HCl, pH

8.0. Washing entails full resuspension, preferably using a tissue homogenizer, followed by centrifugation at 8000x g, the inclusion bodies containing CpcE/CpcF will be in the pellet fraction.

4. The inclusion body proteins are solubi-lized with 9.0 M urea-HCl, pH 1.9, 1 mM DTT. The concentrations of each protein should be determined spec-trophotometrically using the e280 nm for each protein (calculated from the Trp [e = 5540 M-1cm-1] and Tyr [e = 1480 M-1cm-1] content of the proteins) (54).

5. This estimate should be compared with the staining intensities of diluted aliquots of each urea-solubilized protein on SDS-PAGE. The e280 nm for Synechococcus sp. PCC 7002 CpcE and CpcF under denaturing conditions are 35 640 M-1cm-1 and 20220 M-1cm-1, respectively (22).

6. These proteins should be mixed in a 1:1 molar ratio at a concentration of 0.15 to 0.3 mg/mL prior to renatura-tion.

Several methods have been used successfully to renature these proteins. A concentrated mixture can be diluted approximately 1:10 with 50 mM Tris-HCl, 75 mM NaCl, pH 8.0; the dilution is followed by extensive dialysis against the same buffer at 4°C. This procedure yielded renatured het-erodimeric CpcECpcF, but direct dialysis of the diluted proteins in 9.0 M urea against the same Tris-NaCl buffer produced similar results. In both cases, the yield of renatured CpcECpcF was approximately 50%. The extinction coefficients for native CpcE and CpcF were calculated (from the Trp and Tyr content of each protein) to be 38 440 M-1cm-1 and 21060 M-1cm-1, respectively. After filter sterilization through a 0.2 pm membrane to prevent microbial growth, these proteins were stable for weeks at 4°C. Although other purification procedures have been utilized for preparations of proteins for more rigorous kinetic analyses (21), the procedure described above yielded a preparation of enzyme with high activity.

4.2.2. Bilin Donors

PEB and PCB can be cleaved from holo-phycobiliproteins and purified as described elsewhere in this volume (see Chapter 8) and in References 2 and 22. There is presently no reported method for the purification of the precursor of peptide-linked PUB or of the doubly-linked forms of PEB and PUB. However, it has been shown that CpcECpcF from Synechococcus sp. PCC 7002 (13) and Anabaena sp. PCC 7120 (C.F. Chan, W.M. Schluchter, and A.N. Glazer, unpublished results) will transfer the bilin from a holo-a-PC sub-

Figure 5. Bilin addition assays with Anabaena sp. PCC 7120 apo-a-PC resin. Assay conditions were as follows. Approximately 300 ^L of settled resin (containing Anabaena sp. PCC 7120 apo-a-PC subunit covalently attached as described in Reference 22 was in a 1.5-mL microcentrifuge tube containing 0.8 mL of reaction assay buffer (50 mM Tris-HCl, pH 8.0, 75 mM NaCl, 1 mM MgCl2, 1 mM Na pyrophosphate, 1 mM thioglycollate). The enzyme to be tested was Anabaena sp. PCC 7120 CpcECpcF (overproduced and purified as described in this chapter; W.M. Schluchter, C. Chan, and A.N. Glazer, manuscript in preparation). In assays where the enzyme was added (+CpcEF), Anabaena sp. PCC 7120 CpcECpcF were present at 0.25 ^M. In control assays, the same volume of reaction assay buffer was added in place of CpcECpcF (-CpcEF). The reaction was initiated by the addition of the bilin donor. After incubation at 37°C in the dark for 1 hour, the resin was washed extensively as described in the text to remove any remaining donor bilin. The fluorescence emission of the resin present in each assay was measured at 640 nm because this is the peak of fluorescence emission for the native holo-a-PC. The donor bilin was purified PCB (11.6 ^M; labeled as PCB), purified holophycocyanin from Anabaena sp. PCC 7120 (0.92 ^M; labeled as 7120 PC), or purified holophy-cocyanin from Synechococcus sp. PCC 7002 (1.0 ^M; labeled as 7002 PC). The Anabaena sp. PCC 7120 CpcECpcF lyase catalyzed the addition of free PCB to Anabaena sp. PCC 7120 apo-a-PC. However, this enzyme also catalyzed the reverse reaction by transferring bilin from the a-PC subunit (purified either from Anabaena sp. PCC 7120 or from Synechococcus sp. PCC 7002; W.M. Schluchter, C. Chan, and A.N. Glazer, unpublished results).

Figure 5. Bilin addition assays with Anabaena sp. PCC 7120 apo-a-PC resin. Assay conditions were as follows. Approximately 300 ^L of settled resin (containing Anabaena sp. PCC 7120 apo-a-PC subunit covalently attached as described in Reference 22 was in a 1.5-mL microcentrifuge tube containing 0.8 mL of reaction assay buffer (50 mM Tris-HCl, pH 8.0, 75 mM NaCl, 1 mM MgCl2, 1 mM Na pyrophosphate, 1 mM thioglycollate). The enzyme to be tested was Anabaena sp. PCC 7120 CpcECpcF (overproduced and purified as described in this chapter; W.M. Schluchter, C. Chan, and A.N. Glazer, manuscript in preparation). In assays where the enzyme was added (+CpcEF), Anabaena sp. PCC 7120 CpcECpcF were present at 0.25 ^M. In control assays, the same volume of reaction assay buffer was added in place of CpcECpcF (-CpcEF). The reaction was initiated by the addition of the bilin donor. After incubation at 37°C in the dark for 1 hour, the resin was washed extensively as described in the text to remove any remaining donor bilin. The fluorescence emission of the resin present in each assay was measured at 640 nm because this is the peak of fluorescence emission for the native holo-a-PC. The donor bilin was purified PCB (11.6 ^M; labeled as PCB), purified holophycocyanin from Anabaena sp. PCC 7120 (0.92 ^M; labeled as 7120 PC), or purified holophy-cocyanin from Synechococcus sp. PCC 7002 (1.0 ^M; labeled as 7002 PC). The Anabaena sp. PCC 7120 CpcECpcF lyase catalyzed the addition of free PCB to Anabaena sp. PCC 7120 apo-a-PC. However, this enzyme also catalyzed the reverse reaction by transferring bilin from the a-PC subunit (purified either from Anabaena sp. PCC 7120 or from Synechococcus sp. PCC 7002; W.M. Schluchter, C. Chan, and A.N. Glazer, unpublished results).

unit to an apo-a-PC subunit. It is unknown whether all lyases have this transfer activity. However, it is possible that many of these enzymes also serve as repair enzymes or as part of the phycobiliprotein degradation pathway under nutrient starvation conditions (16).

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