NP - Protein examples of purification protocols
Sepaharose purification of full-length SARS NP
(Luo Biochem 2004 pmid: 15323569) E coli BL21(DE3) cells transformed with pET25b-N were grown at 37°C in LB medium to an OD600 of 0.4-0.6 and inducedwith 0.4 mM IPTG for 3.5 h. Cells were harvested by centrifugation and the cell pellets were resuspended and lysed by sonication in buffer A (50 mM Tris-HCl, 2 mM EDTA at pH 7.4) on ice. The cell lysate was centrifuged at 27000g for 20 min at 4°C, and
the supernatant was dialyzed into bufferB (10 mM NaH2PO4, 1mM EDTA at pH 7.4), and was then applied to an SP-Sepharose high-performance column (Pharmacia), which was pre-equilibrated with bufferB. The column was washed with 3 bed volumes of bufferB and then eluted by a linear gradient of NaCl from 0 to 0.8 M in bufferB. The purified protein was dialyzed into bufferB at 4°C. The protein concentration was determined accordingto the method of Edelhoch (14).
Full length protein
(2004 pmid: 15358143) Bacterial expression; His-tag pQE30 vector Qiagen; no gel presented
(2017 pmid: 28720894) Bacterial expression - GST and His-tagged; vector pET32c;
Transformed Escherichia coli BL-21 were grown in 125 ml of LB medium to late exponential phase and after inducing protein expression by addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside, cells were grown at 37 °C or 20 °C for 4 h or 16 h, respectively. Bacteria were harvested, resuspended in 4 ml lysis buffer (PBS, 5 mM DTT, 1 mg/ml lysozyme, 1 mM PMSF, 10% glycerol, 1% Triton X-100 and complete protease inhibitor (Roche) and lysed by two sonication rounds of 10 sec using a Branson sonicator (Danbury, Connecticut, United States). The bacterial lysates were cleared by centrifugation at 15,000 × g for 10 min at 4 °C and passed through a 0.45 µm filter.
For purification of GST fusion proteins, lysates were incubated with 125 µl of glutathione (GSH) Sepharose (4B, GE Healthcare), which had been pre-washed in PBS. Where indicated, lysates were incubated with 40 mg/ml RNase A (Invitrogen, Carlsbad, CA) for 30 min on ice prior to addition to the GSH Sepharose.
6xHis-tagged proteins were used either directly for pull-down experiments or for the purification of the fusion proteins with nickel Sepharose (6 Fast Flow, GE Healthcare) after incubation in presence or absence of 40 mg/ml RNase A for 30 min on ice. Complete hydrolysis of RNA was verified by sample analysis on an agarose gel followed by nucleic acid staining with Midori Green (GC Biotech, Netherlands) or determination of the A260/A280 ratio through measurement of A260 and A280 using a NanoDrop Spectrophotometer (Implen, Germany)54–56.
no coomassie!!!
(pmid: 16979208) Baculo; exhibits RNA binding activity
Deletion constructs
(pmid: 16971454) RNA binding domain - in Bacteria BL21; Analysis of the RNA interaction domains. Alanine substitution of either Arg-76 or Tyr-94 in the N-terminal domain of IBV N protein led to a significant decrease in its RNA-binding activity and a total loss of the infectivity of the viral RNA to Vero cells.
(pmid: 17379242) Bacterial expression: Protein dimerizes, forms oligomers with RNA, helical binding of RNA
(pmid: 19052082) Bacterial expression: all disordered regions are also capable of binding to RNA. Constructs containing multiple RNA-binding regions showed Hill coefficients greater than 1, suggesting that the N protein binds to RNA cooperatively.
!! largest construct 45-365 runs to small on the gel - degradation?? !!