TITLE: Purification of ACOD1 expressed in E. coli
AUTHORS: Konrad Büssow

[DESCRIPTION]
This protocol describes the purification of the human enzyme cis-aconitate decarboxylase (ACOD1) from recombinant E. coli cells. 

[BEFORE_START]
The expression plasmid pCAD29 (pCAD29_hIRG1_4-461_pvp008, Addgene #124843) is used. It contains the coding sequence of human ACOD1, amino acids 4-461, with an N-terminal Streptag and TEV protease cleavage site. The plasmid has a T7 promoter, which is induced by autoinduction here. The method and the plasmid have been described by Chen et al. (F. Chen et al., Crystal structure of cis-aconitate decarboxylase reveals the impact of naturally occurring human mutations on itaconate synthesis. 2019. PNAS 116, 20644-20654).
The ACOD1 protein is purified from the cell lysate using Strep-Tactin XT affinity chromatography. A buffer containing 50 mM biotin is used for elution. Attaching the elution buffer with small size tubing to the chromatography system saves costly biotin. The volume for washing the pump with elution buffer can also be minimized, e.g. 5 ml is sufficient on an ÄKTAprime system.
For a good overexpression of the enzyme, it is important that aeration of the autoinduction cultures is sufficient and that the E. coli cells grow to high density. Polyethyleneimine (PEI) is added to the cell lysates for precipitation of nucleic acids. This leads to a better separation of insoluble material by centrifugation and seems to improve reproducibility and yield of the process. A yield of 5-10 mg can be expected.

[STEPS]
SECTION: Protein expression
2. Inoculate 20 ml MDAG-135 containing 100 mg/L kanamycin and chloramphenicol with a colony and shake over night at 37°C.
SECTION: Protein expression
3. Prepare 4×1 L ZYM-5052 medium with 100 mg/L kanamycin and 34 mg/L chloramphenicol in 2.8 L baffled Fernbach flasks. Inoculate each Fernbach flask with 4 ml overnight culture and shake 24 h at 130 rpm and 25°C. Ensure sufficient aeration of the flasks, do not close them too tightly.
SECTION: Protein expression
4. Check the OD600 of the culture, 12-14 is expected. Dilute the cells 1:20 in water before measurement. Harvest the cells by centrifugation for 20 min at 5000 rpm. Discard the supernatant. Weigh the cell pellets. Up to 100 g wet cell mass is expected. Using a silicone spatula, transfer the cells to a 500 ml beaker. Rinse the centrifugation flasks and the spatula with Wash Buffer. Add the same volume of buffer as the cell volume. Place the beaker on ice. Resuspend the cells with a magnetic stirrer in a cold room. Transfer the cell suspension to 50 ml polypropylene tubes, freeze in liquid N2 and store at -80°C.
SECTION: Protein expression
1. Transform BL21(DE3) CodonPlus-RIL E. coli cells with the plasmid pCAD29. Plate the cells on a SOC agar plate containing 30 mg/L kanamycin and 34 mg/L chloramphenicol and incubate at 37°C overnight.
SECTION: Preparations for purification
5. Prepare a 4 ml Strep-Tactin XT column. Use an empty YMC ECO15/200M0V or similar column. Prepare buffers and 10% PEI.
SECTION: Cell lysis
6. Thaw up to 100 g cell suspension in warm water. Transfer to a 500 ml beaker before thawing is complete. Mix the suspension with a magnetic stirrer. Place on ice when thawing is complete.
SECTION: Cell lysis
7. Increase the volume to 250 ml with Wash Buffer. Lyse the cells by sonication or with a high-pressure homogenizer.
SECTION: Cell lysis
8. Sonication with a Bandlin Sonoplus 2000 Sonifier and a TT13FZ probe:
SECTION: Cell lysis
8.1. Place the beaker with the cells on ice. Immerse the probe half way, about 2 cm, into the cell suspension. Set the amplitude to 58%. For 30 min, run cycles of 1 s sonfication and 9 s pause.
SECTION: Cell lysis
9. Remove 2 µl lysate, mix with 8 µl H2O and 4 µl 4×SDS-PAGE sample buffer
SECTION: Clearing the lysate
10. Add 13 ml 10% PEI per 100 g cells (add less PEI for less cells). Mix, place on ice
SECTION: Clearing the lysate
11. Centrifuge for 45 min at 4°C and 16,500 rpm or higher speed. Repeat if supernatants are not clear. Collect the supernatants.
SECTION: Clearing the lysate
12. Remove 2 µl lysate, mix with 8 µl H2O and 4 µl 4×SDS-PAGE sample buffer.
SECTION: Clearing the lysate
13. Using a 50 ml syringe, filter the supernatant through 0.45 µm syringe filters.
SECTION: Strep-Tactin chromatography
14. Chromatography can be done at room temperature with an ÄKTAPrime Plus. Mount the 4 ml StrepTactin XT column on an Äkta chromatography system. Equilibrate the column with Wash Buffer
SECTION: Strep-Tactin chromatography
15. Apply the cleared cell lysate to the column at 3 ml/min. Place the lysate bottle on ice.
SECTION: Strep-Tactin chromatography
16. Wash the column with 30 ml Wash Buffer at 3 ml/min.
SECTION: Strep-Tactin chromatography
17. Equilibrate the pump with 5 ml Elution Buffer. Elute the protein with 15 ml Elution Buffer at 1 ml/min, collecting 2 ml fractions.
SECTION: Strep-Tactin chromatography
18. Analyse the lysate aliquots, the flow through and the elution fractions by SDS-PAGE. Determine the protein concentration in the fractions by A280 measurement.
SECTION: TEV digest
19. Pool the peak fractions, add 1 mg TEV protease and mix gently. 
Incubate in a refrigerator overnight.
SECTION: Size exclusion chromatography and concentration
20. Transfer the protein into 2 ml microcentrifuge tubes and centrifuge for 30 min at maximum speed. Pool the supernatants.
SECTION: Size exclusion chromatography and concentration
21. Compare the protein size before and after TEV by SDS-PAGE, loading 100 ng protein.
SECTION: Size exclusion chromatography and concentration
22. Equilibrate a Superdex 200 26/60 column with GF Buffer. Apply the protein to the column with a 10 ml sample loop. Elute the protein at 2.5 ml/min and collect 5 ml eluate fractions.
SECTION: Size exclusion chromatography and concentration
23. Analyse peak fractions by SDS-PAGE. Pool peak fractions and add TCEP to 1 mM.
SECTION: Size exclusion chromatography and concentration
24. Concentrate the protein with a Vivaspin 20, MWCO 30,000 concentrator (Sartorius), as needed. Aliquot the protein in 200 µl PCR tubes, freeze in liquid nitrogen and store at -80°C.