N0P constructs comprising full-length PNTD were generated by purifying a heterodimeric N1–525P1–304 complex with a TEV (tobacco etch virus) cleavage site between the two proteins, similar to the production of the shorter P1–50N1–525 described previously (20), or by mixing TEV-cleaved P1–50N1–525 with P1–304 at equal concentrations and subsequent purification of the P1–304N1–525 complex by size exclusion chromatography (SEC; fig. S3). TEV cleavage was performed before a final SEC (Superdex 200, GE Healthcare) in NMR buffer [50 mM Na-phosphate (pH 6), 150 mM NaCl, and 2 mM dithiothreitol (DTT)]. P1–304 was cloned into pET41c(+) between the Nde I and Xho I cleavage sites, where the Xho I site was ligated with a cleaved Sal I site of the insert, yielding a construct with a C-terminal 8His-tag. P1–304 was expressed in Escherichia coli RosettaTM (λDE3)/pRARE (Novagen) overnight at 20°C after induction at an optical density of 0.6 with 1 mM isopropyl-β-d-thiogalactopyranoside. Cells were lysed by sonication and subjected to standard Ni purification in 20 mM tris (pH 8) and 150 mM NaCl. The protein was eluted from the beads in 20 mM tris (pH 8), 150 mM NaCl, and 400 mM imidazole and was then concentrated and subjected to SEC (Superdex 200) in NMR buffer. The HELL mutation was inserted into P by site-directed mutagenesis, and P1–304,HELL→AAAA was expressed and purified the same way. Shorter P constructs (P1–100, P1–160, P140–304, and N1–261) were cloned into pet41c(+) between Nde I and Xho I sites and were subjected to the same expression and purification procedure; the final SEC step was conducted on a Superdex 75 (GE Healthcare) column. N1–525P1–304 was cloned in two steps, first inserting N1–525-TEVsite between the Nde I and Not I cleavage sites of pET41c(+), followed by insertion of P1–304 between the Not I and Xho I sites into the resulting vector. The Xho I site was ligated with a Sal I site of the P1–304 insert. Expression of unlabeled protein was performed in LB medium. Protein labeled for NMR (15N and 13C) was expressed under the same conditions in M9 minimal medium. For deuterated protein (N1–261), the M9 medium was made in D2O.

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



Q&A
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.