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초록

Source: Lopez Jr., M. et al. Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase. J. Vis. Exp. (2017)

This video demonstrates the procedure for purifying viral integrase from bacterial nuclease contamination using heparin Sepharose column chromatography followed by SDS-PAGE analysis to confirm protein purity.

프로토콜

1. Heparin affinity chromatography
   1. Prepare 250 mL Buffer C (50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7.5, 10 mM dithiothreitol (DTT), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 500 μM phenylmethylsulfonyl fluoride (PMSF)) and 250 mL Buffer D (50 mM Tris-HCl, pH 7.5, 10 mM DTT, 0.1 mM EDTA, 500 μM PMSF, 1 M NaCl).
      NOTE: In this protocol, the fast protein liquid chromatography (FPLC) system, buffers, and sample are kept in a cold room at 4 ˚C.
      1. Sterile filter Buffers C and D with 0.2 μm disposable filter units. Depending on the FPLC system, wash pump A and pump B with Buffer C and Buffer D, respectively. Flow 80% Buffer C and 20% Buffer D through the system until the conductivity and ultraviolet (UV) readings stabilize. The maximum flow rate will depend on the instrument; we routinely use a flow rate of 5 mL/min with a maximum pressure limit of 1.0 MPa.
   2. Prepare an 80 mm long, 5 mm diameter FPLC column with 1 mL of heparin Sepharose resin (maximum binding capacity 2.0 mg of bovine antithrombin III per mL resin; maximum pressure 1.4 MPa). The column may be prepared the day before purification and stored at 4 °C. Connect the column to the FPLC instrument.
      1. Equilibrate the column with 20 mL of 80% Buffer C and 20% Buffer D (200 mM NaCl) at a flow rate of 0.5 mL/min with a maximum pressure limit of 1 MPa. The instrument should collect real-time data on conductivity and UV absorbance at 280 nm (A₂₈₀). The conductivity and UV readings should stabilize. If these readings have not stabilized, continue to flow buffers through the column until they are stable.
   3. Reduce the NaCl concentration of the prototype foamy virus integrase (PFV IN) sample to 200 mM by adding 1.5 volumes Buffer C. For example, add 15 mL of Buffer C to 10 mL PFV IN. Our total volume is typically ~25 mL.
   4. Load the diluted PFV IN sample onto the heparin Sepharose column at a flow rate of 0.5 mL/min with a maximum pressure limit of 1.0 MPa. The flow rate and column pressure remain the same throughout the purification. Collect the flow-through in a 50 mL conical tube.
      1. Wash the column with 20 mL of 80% Buffer C and 20% Buffer D (200 mM NaCl), collecting the wash in a 50 mL conical tube. Elute the column with a 30 mL linear gradient of 20% to 100% Buffer D (200 mM to 1 M NaCl).
      2. Wash the column with 5 mL of 100% Buffer D. Collect the gradient and final wash in 0.37 mL fractions.
   5. Analyze the load, flow-through, wash, and fractions by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). PFV IN, following cleavage of the hexahistidine tag, has a molecular weight of 44,394 Da, and the extinction coefficient remains 59,360 cm^-1 M^-1 without the hexahistidine tag. Store all fractions at 4 ˚C until the completion of a nuclease assay.

재료

이 논문에 사용된 재료 목록

이름	회사	카탈로그 번호	댓글
IPTG (Isopropyl β-D-1-thiogalactopyranoside)	Denville Scientific	CI8280
Tris Ultra Pure	Gojira Fine Chemicals	UTS1003
NaCl	P212121	RP-S23020
PMSF (Phenylmethanesulfonyl fluoride)	Amresco	754
DTT (Dithiothreitol)	P212121	SV-DTT
UltraPure EDTA	Invitrogen/Gibco	15575
Heparin Sepharose 6 Fast Flow	GE Healthcare Life Sciences	17-0998-01