Procedure 5 Purification of His Tagged UROS from E coli

1. Bacterial growth: The bacteria are grown from a starter culture in 2-L baffled flasks containing 1 L of LB media (with appropriate antibiotics) at 37°C with vigorous shaking until an A600 = 0.6 is reached, at which point isopropyl-P-D-thiogalactoside (IPTG) is added to a final concentration of 0.4 mM, and the cells are grown for another 2 hours.

2. Harvesting and cell lysis: The bacteria are collected by centrifugation (10 000x g at 4°C). The bacterial pellet is resus-pended in 10 mL of binding buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The bacterial suspension is sonicated as described in Procedure 1, and the solution is cen-trifuged (10 000x g at 4°C) to remove the cellular debris.

3. His-bind column: The His-tag sequence of the fusion protein can bind to divalent metal cations such as Co2+ and Ni2+ immobilized on to His-bind resin (Novagen, Madison, WI, USA; however, many suppliers make different forms of metal chelate resin and readers are encouraged to browse the multitude of catalogues available). After unbound proteins are washed away, the His-tagged protein is eluted with imidazole. The resin (poured into a small column, 1 x 2.5 cm) is initially prepared by rinsing with 15 mL of water, charged with 25 mL of a 50 mM divalent cation solution (normally Ni2+) (charge buffer), and equilibrated with 15 mL binding buffer. The supernatant is loaded onto the charged His-bind column. The column is washed with 10 column volumes of binding buffer, 6 column volumes of wash buffer (100 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), and finally the protein is eluted in 6 column volumes of elution buffer (400 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). The protein eluting from the column can be detected by the use of the Bio-Rad protein assay and SDS-PAGE.

4. Storage: Fractions containing the modified UROS are pooled and desalted by passing through a PD-10 column, previously equilibrated in 50 mM Tris-

HCl, pH 7.8. The protein is lyo-philized and is stable in this form for up to 1 year. In comparison to some of the other enzymes described in this chapter, UROS is poorly expressed, and a yield of about 2 mg/L of culture is normally achieved.

6.2. Enzymatic Preparation of Uroporphyrinogen III

Uroporphyrinogen III can be synthesized in vitro using PBG and purified PBGD and UROS. The reaction can be undertaken in a range of buffers between pH 7.5 and 9.0, although the uropor-phyrinogen III is generally more stable at the higher pH values. To prevent oxidation of the product, the buffers are normally thoroughly degassed by freeze—thawing under a vacuum of less than 1 mbar. For efficient transformation of PBG into uro-porphyrinogen III, the reaction mixture should contain PBGD at 10 pg/mL, UROS at 2 pg/mL, and PBG at 100 pM. The reaction is effectively quantitative, thus producing uroporphyrinogen III at a concentration approaching 25 pM. This can be verified by taking 50 pL of the incubation, mixing with 950 pL of 1 N HCl, and leaving under a bright light for 20 minutes. After centrifugation in an Eppendorf model microfuge at 13 000 rpm for 5 minutes, the absorbance of the solution at 405 nm can be measured, and the concentration of porphyrin can be determined using the extinction coefficient of 5.48 x 105 M-1 L.

So long as the enzymatic incubation is kept in an anaerobic environment under reduced light, the uroporphyrinogen III is stable for several hours. The solution should appear colorless, but if it starts to turn pink then this is diagnostic of the solution starting to oxidize. To isolate the uroporphyrinogen III from the incubation (i.e., to remove the enzymes from the reac tion mixture) the solution can be filtrated in an ultrafiltration unit fitted with a PM-10 membrane. The filtrate should be kept under argon to help prevent any oxidation. The yield of uroporphyrinogen III from PBG is normally in excess of 95%.

The uroporphyrinogen I isomer can also be synthesized by this method simply by omitting UROS from the incubation.

7. SYNTHESIS OF PRECORRIN-2 (DIHYDROSIROHYDRO-CHLORIN), SIROHYDRO-CHLORIN, AND SIROHEME

Enzymatic transformations of uropor-phyrinogen III into precorrin-2 are dependent upon the presence of the enzyme uro-porphyrinogen III methyltransferase (Figure 5), which requires S-adenosyl-L-methionine (SAM) as a methyl donor (3). There are a number of sources of this enzyme including Pseudomonas denitrifi-cans, Bacillus megaterium, and Bacillus stearothermophilus. The CysG enzyme from both E. coli and Salmonella typhimurium can also be used, although CysG is, in fact, a multifunctional enzyme responsible for the conversion of precorrin-2 into siroheme (30). However, in the presence of only SAM and uroporphyrinogen III, the enzyme will effectively transform uropor-phyrinogen III into precorrin-2. The uro-porphyrinogen methyltransferases are normally homodimers with a subunit molecular mass of about 30 kDa, while the CysG proteins, which are also homodimers, have a subunit molecular mass of 50 kDa.

7.1. Purification of Uroporphyrinogen Methyltransferases

Although the uroporphyrinogen methyl-transferases can be purified from recombinant sources, the preparations are often laborious and in low yields. We have favored

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