친화성 크로마토그래피를 이용한 바이러스 인테그레이스의 정제

초록

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

This video demonstrates the purification of polyhistidine-tagged viral integrase using nickel affinity chromatography. The protocol uses imidazole gradient elution to selectively isolate the integrase based on its affinity for nickel-charged resin.

프로토콜

1. Nickel Affinity Chromatography

1. Thaw one bacterial pellet on ice.
   NOTE: This will take significant time (approximately 2 - 3 h). When the pellet has thawed to a cell slurry, add an additional 500 μM Phenylmethanesulfonyl Fluoride (PMSF) (100 mM stock solution).
2. Keep the 50 mL tube of bacteria slurry on ice. Sonicate the bacteria at 30% amplitude for 30 s (1 s on, 1 s off) per pellet derived from 1 L culture using a sonicator with a 0.5 inch diameter bio horn with a 0.125 inch diameter tapered microtip, a frequency of 20 kHz and maximum power of 400 W.
3. Transfer the cell sample to a cold ultracentrifuge tube. Use polycarbonate bottle assemblies (25 mm diameter, 89 mm height) compatible with a Type 60 Ti fixed angle rotor. Alternative tubes and rotors may be used if the same gravitational force is achieved. Spin 120,000 x g for 60 min at 4 °C. There should be an obvious pellet. The supernatant may have a yellow color.
4. Transfer the supernatant by pouring or pipetting to a cold 50 mL conical tube. Note the volume; this volume should be approximately the volume of resuspension buffer used prior to snap freezing. If the supernatant is viscous, it may be contaminated by bacterial DNA which could clog the chromatography column. In this case, repeat the ultracentrifugation to pellet the bacterial DNA.
5. Prepare 500 mL Buffer A (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 500 μM PMSF) and 500 mL Buffer B (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 500 μM PMSF, 200 mM imidazole).
   NOTE: In this protocol, keep the fast protein liquid chromatography (FPLC) system, all buffers and the sample at 4 °C in a cold room.
   1. Sterile filter Buffers A and B with 0.2 μm disposable filter units. Depending on the FPLC system, wash pump A and pump B with Buffer A and Buffer B, respectively. Flow 90% Buffer A and 10% Buffer B through the system until the conductivity and ultraviolet (UV) readings stabilize. The maximum flow rate will depend on the instrument; use a flow rate of 5 mL/min with a maximum pressure limit of 1.0 MPa.
6. Prepare a 110 mm long, 5 mm diameter FPLC column with 1.5 mL nickel-charged resin (maximum binding capacity 50 mg/mL, maximum pressure 1 MPa). The column may be prepared the day before purification and stored at 4 ˚C.
7. Connect the column to the FPLC instrument. Equilibrate the column with 20 mL of 90% Buffer A and 10% Buffer B (20 mM imidazole) at a flow rate of 0.5 mL/min with a maximum pressure limit of 0.5 MPa. The instrument should be collecting real time data of 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.
8. Load the protein sample (~10 mL per pellet) to the column at a flow rate of 0.15 mL/min with a maximum pressure limit of 0.5 MPa. The flow rate and column pressure remain the same throughout the purification. Collect the flow through in a 50 mL tube.
   1. Wash the column with 20 mL 90% Buffer A and 10% Buffer B. Collect the wash in a 50 mL tube. Elute the proteins with a 20 mL linear gradient of 10% to 100% Buffer B (20 mM to 200 mM imidazole).
   2. Finally, wash the column with 5 mL 100% Buffer B (200 mM imidazole). Collect the gradient and final wash in 0.27 mL fractions.
9. Combine 15 μL of 2x SDS-PAGE sample buffer with 15 μL of the flow through, wash, and peak A₂₈₀ fractions. Boil the samples for 3 min.
   1. Prepare two 10% SDS-PAGE gels. Load 10 μL of each sample to the gels and 4 μL of prestained protein standards. Run, stain, and analyze the gels.
   2. Combine fractions that appear to contain nearly pure prototype foamy virus integrase (PFV IN) based on visual inspection. Measure the volume by pipetting, typically 8 - 10 mL.
   3. Using a spectrophotometer, measure the A₂₈₀ of the combined fractions and calculate the total amount of PFV IN protein; the typical yield is ~10 mg per liter of induced culture. The extinction coefficient of PFV IN with the hexahistidine tag is 59360 cm^-1 M^-1.

재료

이 논문에 사용된 재료 목록

이름	회사	카탈로그 번호	댓글
BL21/DE3 Rosetta E. coli	EMD Millipore	70956-4
Imidazole	Sigma-Aldrich	I0250
Ni-NTA Superflow	Qiagen	30430