TITLE: Purification of recombinant Low Density Lipoprotein Receptor Related Protein Associated Protein 1 (LRPAP1, RAP) from Escherichia coli
AUTHORS: Patricia Yuste-Checa, Silvia Gärtner, F Ulrich Hartl

[DESCRIPTION]
This protocol details how to efficiently purify LDL Receptor Related Protein Associated Protein 1 (LRPAP1 or RAP) from Escherichia coli.

[STEPS]
SECTION: LRPAP1 express4ion
1. Thaw RbCl-competent Escherichia coli Bl21 cells (DE3) on ice.
SECTION: LRPAP1 express4ion
2. Add 1 µL of pQTEV-LRPAP1 plasmid without the signal peptide (1-35 amino acids) and incubate 30 min on ice.

SECTION: LRPAP1 express4ion
3. Heat shock 45 s at 42 °C.

SECTION: LRPAP1 express4ion
4. Incubate on ice 2 min, then add 850 µL Lysogeny broth (LB) or Super Optimal broth with Catabolite repression (SOC) medium.

SECTION: LRPAP1 express4ion
5. Shake for 60 min at 37 °C.

SECTION: LRPAP1 express4ion
6. Centrifuge for 3000 x g, 5 min and remove most of the supernatant.

SECTION: LRPAP1 express4ion
7. Resuspend the pellet with the remaining supernatant and plate the bacteria on LB /Ampicillin agar plates and incubate 60 min at 37 °C.

SECTION: LRPAP1 express4ion
8. Prepare preculture: Scrap all colonies with the scraper and inoculate 25-50 mL LB/Ampicillin. Shake at 37 °C for 4-6 h.

SECTION: LRPAP1 express4ion
9. Measure OD600 of the preculture and inoculate 6 L of LB media to an OD600 = 0.05. 

SECTION: LRPAP1 express4ion
10. Shake flasks at 37 °C until approx. OD600 = 0.5-0.8. (2-4 h).

SECTION: LRPAP1 express4ion
11. Add isopropyl β-D-1-thiogalactopyranoside (IPTG) at final concentration of 1 millimolar (mM).

SECTION: LRPAP1 express4ion
12. Shake flasks 60 min at 22 °C.

SECTION: LRPAP1 express4ion
13. Centrifuge bacterial culture at 4000 rpm, 60 min. Discard supernatant.

SECTION: LRPAP1 express4ion
14. Resuspend each pellet with Lysis buffer (20 mL/1L bacteria) supplemented with Complete EDTA-free protease inhibitor cocktail (Merck). Flash-freeze in liquid nitrogen for storage at -80 °C.

SECTION: Lysis
15. Thaw the cell pellets in a water bath at 22 °C and add lysis buffer (final volume 200 mL lysis buffer/ 6L bacteria) supplemented with Complete EDTA-free protease inhibitor cocktail (Merck) and Sm DNase 50 1584.

SECTION: Lysis
16. Add 1 1629 lysozyme and incubate gently shaking for 30 min at 4 °C.

SECTION: Lysis
17. Sonicate lysate on ice, 8 cycles 20 s ON, 30 s OFF.

SECTION: Lysis
18. Centrifuge lysate at 40000 rpm, 45 min, 4 °C.

SECTION: Ni-NTA chromatography
19. Equilibrate the Ni-NTA column with 10 column volumes (CV, 20 mL) Lysis buffer.
SECTION: Ni-NTA chromatography
20. Load lysate supernatant to the Ni-NTA column.
SECTION: Ni-NTA chromatography
21. Wash the Ni-NTA column with 10 CV Lysis buffer.
SECTION: Ni-NTA chromatography
22. Elute His7-TEV-RAP with 5 CV 100% High salt buffer and collect elution fractions.
SECTION: Ni-NTA chromatography
23. Analyze eluted fraction by SDS-PAGE and Coomassie blue staining.
 

SECTION: Size exclusion chromatography
30. Load RAP-containing fractions onto a Superdex-200 column previously equilibrated with SEC buffer.
SECTION: Size exclusion chromatography
31. Analyze eluted fractions by SDS-PAGE and Coomassie blue staining.
 

 

SECTION: Size exclusion chromatography
32. Pool fractions containing RAP aliquot and flash-freeze in liquid nitrogen for storage at -80 °C.
 


SECTION: Desalting
24. In order to reduce the salt concentration, load the eluted protein onto a HiPrep 26/10 desalting column equilibrated with the Low salt buffer.
SECTION: His-TEV cleavage
25. Collect eluted fraction containing protein and add glycerol at final concentration of 10%, DTT at final concentration of 1 millimolar (mM), EDTA at final concentration of 0.25 millimolar (mM) and His-TEV at final concentration of 93U per mg of protein.

SECTION: His-TEV cleavage
26. Incubate at 4 °C 30 s.

SECTION: Ni-NTA chromatography (Collect flow through)
27. Load the cleavage mixture onto a Ni-NTA column previously equilibrated with Lysis buffer and collect the flow through where the cleaved RAP protein should elute.
SECTION: Ni-NTA chromatography (Collect flow through)
28. Wash the column with 5 CV (CV, 20 mL) of Lysis buffer and collect eluted fractions.
SECTION: Ni-NTA chromatography (Collect flow through)
29. Analyze flow though fractions by SDS-PAGE and Coomassie blue staining.