The cDNA sequence encoding full-length WT p17/PERMIT was cloned into a pET28b(+) vector using Nde I and Sac I restriction sites. The resulting construct (pET28b-p17) produces a 175-residue (19.1 kDa) fusion protein containing a thrombin-cleavable N-terminal 6×His tag, preceding the 155–amino acid p17/PERMIT coding sequence. This plasmid was used as a template for QuikChange site-directed mutagenesis (Agilent) to generate a triple-alanine mutant (pET28b-p17/PERMITR28–30A) of the RYE residues at positions 28 to 30 (R28A, Y29A, and E30A). Both WT and mutant p17/PERMIT constructs were used to transform E. coli BL21(DE3) cells. Cells were grown to ODA595 = 0.8 in 1 liter of LB media, and protein expression was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 4 hours at 37°C. Since >90% of His6-p17/PERMIT and His6-p17/PERMITR28–30A were expressed in the insoluble fraction, a urea-denaturating/on-column refolding purification was used for both constructs. Briefly, cells were pelleted by centrifugation for 20 min at 4°C (10,000g) and resuspended in 40 ml of Lysis Buffer A, containing 20 mM tris (pH 7.4), 500 mM NaCl, 3 mM beta-mercaptoethanol (βME), deoxyribonuclease I (DNase I) (2.5 μg/ml) (#D5025; Sigma-Aldrich), 1× phenylmethylsulfonyl fluoride (PMSF), and one PI tablet (#A32965; Pierce). Cells were lysed by sonication and centrifuged at 4°C for 30 min (15,000g) to pellet the His6-p17–containing insoluble fraction. The supernatant was discarded, and the pellet was resuspended in 40 ml of Lysis/Wash Buffer B [6 M urea, 20 mM tris (pH 7.4), 500 mM NaCl, 20 mM imidazole 3 mM βME, 1× PMSF, and 1× PI tablet]. Following a second round of sonication and centrifugation, the urea lysate was applied to an equilibrated 1-ml HisTrap FF affinity column (GE Healthcare) and the column was washed with 20 volumes (c.v.) of Buffer B. His tag–immobilized p17 was refolded on-column by applying a linear gradient of 6 to 0 M urea over 30 c.v. at a flow rate of 0.5 ml/min. Renatured protein was liberated from the column with a 0 to 100% linear gradient of Elution Buffer C [20 mM tris (pH 7.4), 500 mM NaCl, 500 mM imidazole, 3 mM βME, and 1× PI tablet] over 10 c.v. Eluted fractions were analyzed by SDS-PAGE, and fractions containing His6-p17 at purity >80% were combined, EDTA was added to 500 μM, and fractions were concentrated to ~3 ml using an Amicon Ultra-15 (10,000 molecular weight cut off) centrifugal concentrator (Millipore). Contaminating proteins were removed by size exclusion chromatography (SEC) by applying the concentrated sample to a preparative-scale HiLoad 26/600 Superdex 75 pg column pre-equilibrated with SEC Buffer D [1× PBS (pH 7.4), 500 μM EDTA, 3 mM βME, and 1× PI tablet]. Isocratic elution at a flow rate of 2.5 mg/ml resulted in a monodisperse peak containing His6-p17/PERMIT (WT and mutant) at >95% purity. Protein concentration was determined by ultraviolet absorbance at 280 nm using the molecular weight (MW) and extinction coefficient (ε) for each construct (His6-p17/PERMIT MW = 19,356 g/mol, ε = 8480 M−1 cm−1; His6-p17/PERMITR28–30A MW = 19,120 g/mol, ε = 8480 M−1 cm−1). A typical purification yielded approximately 2.5 g of purified protein per liter of rich media. Purified protein was stored as 200-μl aliquots at −80°C.

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.