Enterokinase Purification

Protocol

Protein Expression

1, Transform BL21Star(DE3)pRare2pLysS cells with 1731 pT7A3-TurboEnterokinase.

2, Inoculate 200 mL medium with a few bacterial colonies. Incubate this preculture o/n at 37 °C and 180 rpm. Alternatively, obtain the inoculum from a glycerol stock culture.

3, Inoculate 8 × 1000 mL LB/KC medium with 25 mL preculture per flask, i. e. 1 : 40 (v/v). Incubate these main cultures at 37 °C and 180 rpm.

4, As soon as OD600 reaches 1.0 (about 2~4 h), induce enterokinase expression by adding 0.1 to 0.5 mM IPTG to the cultures. Maintain temperature and rpm.

5, After 4 h, harvest cells by centrifugation (5 min at 6000 rpm and 4 °C). Weigh pellet on balance. Pellet can be stored at −20 °C.

IB Solubilization

1, Resuspend pellet in Lysis Buffer at 10~15 mL/g. Freeze and thaw once.

2, Lyse cells by homogenization (~30 to 60 sec) or sonication (3 × 5 min with 50 % duty time and 95 % power, interrupted by at least 5 min chilling on ice).

3, Stir or (shake gently) the lysate for 30 min at rt.

4, Spin down particulate matter (25000 rpm, 20 min, 4 oC) and discard supernatant.

5, Resuspend in 80 ml Lysis Buffer, use homogenizer for efficient resuspending.

6, Spin down IB (25000 rpm, 20 min, 4 oC) and discard supernatant.

7. Repeat step 5 & 6.

8, Resuspend pellet in 80 mL Suspension Buffer, again, use a homogenizer.

9. Spin down IB (25000 rpm, 20 min, 4 oC) and discard supernatant. Weigh IB weight.

IB can be stored at −20 °C.

10, Dissolve 1 g IB in 20 mL Solubilization Buffer I, i.e. at 1 : 20 (w/v).

11, Stir the solution o/n at RT.

12, Spin down undissolved particles (25000 rpm, 20 min, 4 oC).

13, Adjust the pH of the supernatant to 3.5–4.0.

This step is of paramount importance, since a low pH prevents random disulfide bonds from forming prior to refolding!

14, Dialyze the supernatant against Citrate Buffer at RT.

The molecular weight cutoff of the dialysis membrane should be 6–8 kDa. Due to the large supernatant volume (e. g., 400 mL), a dialysis ratio of about 1 : 30 suffices (i. e., 3 × 130 mL supernatant against 3 × 4 L Citrate Buffer). Exchange the buffer twice, which yields exemplary dialysis durations of 4 h, 4 h, and o/n.

(or load to desalting column, use Citrate Buffer).

15, Harvest precipitated protein by centrifugation (25000rpm, 20 min, 4 oC). Pellets may be stored at −20 °C. The total pellet weigh should approximate the value from step 9 (typically 20 g).

16, Dissolve 1 g IB in 10 mL Solubilization Buffer II, i.e. 1 : 10 (w/v). If the solution appears cloudy, spin down particulate matter (25000 g, 20 min, 4 oC). The total volume will be about 200 mL.

Refolding

1, Prepare 4 L Refolding Buffer. 2, Dilute the protein solution dropwise into the Refolding Buffer. The final volume ratio must be at least 1 : 100. In order to minimize the amount of Refolding Buffer required, apply the solution in two pulses separated by an interval of 24 h. For example, if the IB solution volume is 200 mL, prepare 3 × 4 L Refolding Buffer and dilute 2 × 35 mL IB solution into each cup, with one day between the two pulses. Dropwise dilution is achieved by using a burette (or a 5 mL pipette) and stirring the buffer.

3, Incubate the refolding solution for 3–4 d at 16 °C without stirring.

4, Concentrate 4 L refolding solution to about 200 mL by tangential flow on wet ice (molecular weight cutoff 10 kDa).

How to handle the concentrator: (1) Flush the system with 2 L dH2 O, followed by 500 mL Refolding Buffer (without cysteine/cystine). (2) Concentrate the refolding solution to about 100 mL. (3) Rinse the concentrator with 100 mL Refolding Buffer (without cysteine/cystine), which yields a final concentrate volume of 200 mL. (4) Clean the system with 2 L 0.2 m NaOH for 5 min. (5) Store the tubes in 2 L freshly prepared 0.2 m NaOH (adjust 67.5 g 32 % NaOH to 2 L with dH2O).

5, Dialyze the concentrate against Storage Buffer at RT. The molecular weight cutoff of the dialysis membrane should be 6–8 kDa. Due to the large concentrate volume (e. g., 600 mL), a dialysis ratio of about 1 : 20 suffices (i. e., 3 × 200 mL supernatant against 3 × 4 L Storage Buffer). A single buffer exchange leads to dialysis durations of 4 h–o/n or vice versa.

6, Spin down precipitate (25000 rpm, 20 min, 4 °C)

7, Add 3 mM NaN3 to the supernatant and store it at 4 °C for 3 d to ensure complete autoactivation.

Purification

1, Add 20 mL High-NaCl Buffer to 200 mL enterokinase. This step increases the NaCl concentration to about 500 mm, which prevents unspecific interactions between impurities and the resin.

2, Incubate with 6 mL benzamidine sepharose 4 Fast Flow (high sub) resin (equilibrated with Binding Buffer) for 30 min at r t.

3, Collect the flow-through.

4, Wash the resin with 40 mL Binding Buffer.

5, Elute bound enterokinase with 15 mL Elution Buffer 25, 50 and 100. Elution Buffer 25 contains 25 mM benzamidine &c.

6, If SDS-PAGE indicates residual enterokinase in the flow-through, repeat steps 2 to 5 (load the flow-through).

7, Combine all eluates, concentrate them to 80–100 μg mL−1 and exchange the buffer to Cryobuffer. For example, three purification batches will yield about 135 mL combined eluate. (1) Concentrate this volume to 3 mL using two Amicon Ultra-15 Centrifugal Filter Units with a molecular weight cutoff of 10 kDa. (2) Dilute the concentrate tenfold with Storage Buffer and concentrate it again to 3 mL. (3) Repeat step (2) twice. (4) Dilute the final concentrate to 80–100 μg mL−1 with Cryobuffer.

8, Store enterokinase at −80 °C.

Buffers

Lysis Buffer

Tris 50 mm, 3.03 g (for 500 mL), pH 7.
NaCl 500 mM
EDTA 20 mm
Triton X-100 6%

Suspension Buffer
Tris 50 mM 3.03 g (for 500 mL), pH 7.0
EDTA 20 mM 2.92 g

Solubilization Buffer I

Guanidine-HCl 7.5 M, 716.48 g L−1
Tris 50 mM, 6.06 g L−1
β-Mercaptoethanol 100 mM, 6.97 mL L−1
pH (native) ≈9

Citrate Buffer
Citrate 5 mM 4.20 g (for 4 L)
pH 3.5–4.0

Solubilization Buffer II

Guanidine-HCl 7.5 M, 358.24 g (for 500 mL)
Tris 50 mM 3.03 g
pH 4.0–4.5

Refolding Buffer
Arginine 0.5 M 348.4 g (for 4 L)
Tris 50 mM 24.24 g
CaCl2 20 mM 11.80 g
EDTA 1 mM 1.16 g
Cysteine-HCl 5 mM 3.52 g
Cystine 0.5 mM 0.48 g
pH 8.3 at RT

Note: Dissolve cystine in 5 mL 2 N HCl

Storage Buffer
Tris 50 mM 24.24 g (for 4 L)
NaCl 50 mM 11.68 g
pH 7.5

High-NaCl Buffer
Tris 50 mM, 3.03 g (for 100 mL)
NaCl 5M, 29.22 g
pH 7.5

Binding Buffer
Tris 50 mM 3.03 g (for 500 mL)
NaCl 500 mM 14.6 g
pH 7.5

Elution Buffer 100.
Binding Buffer 100 mM 50 mL
Benzamidine-HCl 0.78 g
pH 7.5

Cryobuffer
Tris 50 mm 3.03 g (for 500 mL)
NaCl 50 mm,  1.46 g
Glycerol 10 % (v/v), 50 mL
pH 7.5

Old protocol

1. Transform BL21 Star (DE3)/pRare2pLysS with plasmid

2. Grow cells o/n in 100 ml LB/Kan/Chl.

3. Inoculate 2 L LB/Kan/Chl with 100 ml (1 : 40, v/v) from an o/n preculture.

4. Grow at 37 °C and 200 rpm to OD600 1.6.

5. Induce with 0.1 mM IPTG and grow o/n at 30 to 37 oC.

6. Harvest cells by spining at 6000 rpm.

7. Resuspend cells with 10 ml of lysis buffer per gram of wet cell.

 50 mM Tris-HCl, pH 7.0, 2 % Triton X-100, 500 mM NaCl

8. Lysis by freeze-thaw.

9. Homogenize.

10. Spin at 12000 rpm for 20 min.

12. Dissolve pellet  in Buffer P: 1g pellet in 20 ml buffer

7.5 M guanidine-HCl, 50 mm Tris-HCl, 100 mm β-mercaptoethanol.

13. Adjust pH to 3.5–4,
14. Dialyze the solution against 5 mM citrate pH 3.5–4.0.

15. Spin at 12000 rpm.

16. Dissolve precipitated protein in 7.5 M guanidine-HCl, 50 mM Tris pH 4.0–4.5.

17. Load to Ni column and elute with 300 mM imidazole.

18. Concentrate the protein using spin columns.

19. Refolding:  dropwise dilution of the protein solution into the 100-fold volume
of a buffer containing 0.5 M arginine, 50 mM Tris pH 8.3, 20 mM CaCl2 , 1 mM EDTA, 5 mM cysteine, 0.5 mM cystin.

20. Incubate at 16  °C for 72–96 h.

21. Optional: load the enterokinase concentrate onto a benzamidine sepharose 4 Fast Flow (high sub) column (GE Healthcare). After washing the resin with 50 mm Tris pH 7.5, 500 mm NaCl, correctly folded enterokinase can be eluted with the same buffer supplemented with 25–100 mm benzamidine.

21. Concentrate 20 times of the original volume using 9kDa spin columns

22. Load to a TSK gel filtration column and purify using 50 mm Tris pH 7.5, 50 mm NaCl.
23. Concentrate the eluates to approximately 0.2 mg/ml, add equal volume of 20 % (v/v) glycerol.

24. Store aliquoted protein at −80 °C