The gene encoding the tail fiber of P68 in plasmid pMUH17 was a gift from U. Bläsi from Max F. Perutz laboratories in Vienna (35). The tail fiber protein was purified as described previously (35), with minor changes. The His-tagged tail fiber protein was expressed in Escherichia coli strain BL21 (DE3). After overnight cultivation in LB medium at 37°C and 200 rpm, the preculture was diluted 100× into 1000 ml of fresh LB medium. The culture was grown at 37°C with 200 rpm shaking until an OD600 of 0.7 was reached. The expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside to a final concentration of 1 mM. After 4 hours of protein expression at 37°C and 200 rpm shaking, the culture was centrifuged at 4000g for 30 min. The cell pellet was resuspended in 50 ml of lysis buffer [300 mM NaCl, 40 mM imidazole, 50 mM NaH2PO4 (pH 8.0), 1 mM phenylmethylsulfonyl fluoride (PMSF), and lysozyme (1 mg/ml)], incubated for 1 hour at 4°C, and homogenized by sonication using a Qsonica Q700 sonicator (2-s sonication, 2-s pause, 4-min sonication, 50 W). The cell lysate was centrifuged at 20,000g for 40 min at 4°C and loaded onto a HisTrap column (GE Healthcare) equilibrated with lysis buffer. Most of the impurities were eluted with wash buffer [70 mM imidazole, 300 mM NaCl, 50 mM NaH2PO4 (pH 8.0), and 1 mM PMSF]. His-tagged tail fiber protein was eluted from the column using elution buffer [500 mM imidazole, 300 mM NaCl, 50 mM NaH2PO4 (pH 8.0), and 1 mM PMSF]. The solution containing tail fiber protein was dialyzed against 25 mM tris-HCl (pH 7.5), 150 mM NaCl buffer, followed by separation in a HiLoad 16/600 Superdex 200 pg size exclusion column (GE Healthcare). The tail fiber protein eluted in the void volume of the column. The protein at a concentration of 5 mg/ml in 25 mM tris-HCl (pH 7.5), 150 mM NaCl buffer was used for crystallization screening. Crystals were obtained in a CrystalEX 96-Well conical bottom plate (Hampton Research) by the sitting-drop vapor diffusion method by mixing 0.5 μl of protein solution with 0.5 μl of 200 mM MgCl2, 100 mM Hepes (pH 7.0), and 20% (w/v) PEG 6000 (polyethylene glycol, molecular weight 6000). Hexagonal crystals with dimensions up to 200 μm formed within 30 days.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.