FGF Expression and Purification Protocol

(Dr. Jihun Lee, June 5 2007)

 

 

Expression (1L scale)

 

 

M9 media composition for IL

 

Na2HPO4 (Diabasic) : 12.0gm

KH2PO4 : 3.0gm

NaCl : 0.5gm

NH4Cl : 1gm

ddH2O : 987ml

 

Autoclave

 

 

M9 media additives for 1L

 

20% (w/v) D-glucose : 20ml

1M MgSO4 : 2ml

1% Thiamine : 100 ul

0.5% (w/v) FeCl2 : 150 ul

 

add these additives right before starting culture

 

 

  1. Inoculate single colony to 100ml of M9 media (w/ ampicillin: 0.15mg/ml media) grow culture at 37C, ~270rpm, for ~18hr

 

  1. Pour the overnight culture to 1L M9 media (w/ ampicillin: 0.15mg/ml media) grow culture at 37C for 2~3hr

 

  1. Check the cell O.D: when O.D600 reaches 0.6~0.7, put 1ml of 1M IPTG (final IPTG concentration: 1mM)

 

  1. Change temperature to 28C incubate for 5~6hr

 

  1. Harvest cells by centrifuging at 6,000 rpm for 15min

 

  1. Keep cell pellet at -20C until cell lysis

 

 

Cell lysis

 

Buffer

Composition

Amount for 1L

pH

5mM imidazole

50mM NaPi

500mM NaCl

5mM imidazole

Dibasic (0.5M): 80.95ml and Monobasic (1M): 9.5ml

29.22gm

0.3404gm

 

7.5

 

 

  1. Dissolve cell pellet with [80~100ml of 5mM Imidazole buffer + 80~100ul of 10% tween 80]

 

  1. Break cells with French Press (under 1000 psi pressure)

 

  1. Centrifuge cell lysate at 15,000 rpm for 30min

 

  1. Transfer supernatant into the flask: if supernatant is too viscose, add 50ml~200ml of 5mM Imidazole buffer

 

 

Ni-NTA column purification

 

Ni-NTA buffer

Buffer

Composition

Amount for 1L

pH

5mM imidazole

50mM NaPi

500mM NaCl

5mM imidazole

Dibasic (0.5M): 80.95ml and Monobasic (1M): 9.5ml

29.22gm

0.3404gm

 

7.5

50mM imidazole

50mM NaPi

500mM NaCl

50mM imidazole

 

Dibasic (0.5M): 80.95ml and Monobasic (1M): 9.5ml

29.22gm

3.404gm

 

7.5

250mM imidazole

50mM NaPi

500mM NaCl

250mM imidazole

 

Dibasic (0.5M): 80.95ml and Monobasic (1M): 9.5ml

29.22gm

17.02gm

 

7.5

* Adjust pH with either 10M NaOH or 1M NaOH

 

 

  1. Equilibrate Ni-NTA column (using 20ml of Ni-NTA (nickel-nitrilotriacetic acid) resin) with 5 mM Imidazole buffer

 

  1. Load supernatant of cell lysate on Ni-NTA column

 

  1. Wash with 100ml of 5 mM Imidazole buffer

 

  1. Wash with 100ml of 50mM Imidazole buffer

 

  1. Elute protein with 100~200ml of 250mM Imidazole buffer: usually, protein starts eluting after passing ~15ml of 250mM Imidazole buffer

 

* since protein is eluted within narrow range of fractions, protein may be precipitated immediately after elution (elution becomes cloudy). In this case, dilute with 250mM Imidazole buffer and filter it.

 

  1. Recommended fraction collector setting: 150drops/fraction or 5ml/fraction

 

  1. Based on protein elution profile, take samples from fractions and run 15% SDS-PAGE decide which fractions should be pooled (see Fig.1)

 

  1. Pool the fractions

 

 

 

 

 

 

 


 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1. Typical Ni-NTA column elution profile and SDS-gel of wild-type FGF

 

 

Heparin column purification

 

Heparin Buffers

Buffer

Composition

Amount for 1L

pH

Heparin Buffer

50mM NaPi

500mM NaCl

10mM (NH4)2SO4

2mM DTT

Dibasic (0.5M): 80.95ml and Monobasic (1M): 9.5ml

29.22gm

1.3214gm

0.3084gm

 

7.5

Heparin Elution Buffer

50mM NaPi

2M NaCl

10mM (NH4)2SO4

2mM DTT

Dibasic (0.5M): 80.95ml and Monobasic (1M): 9.5ml

116.88gm

1.3214gm

0.3084gm

 

7.5

 

 

  1. Equilibrate Heparin column (using 20ml of Heparin Sepharose CL-6B affinity resin) with Heparin buffer

 

  1. Load Ni-NTA elution pool on Heparin column

 

  1. Wash with 100ml of Heparin buffer

 

  1. Elute protein with linear gradient of NaCl : using gradient maker, set linear gradient from 0.5 M to 2M NaCl (200ml of Heparin buffer / 200ml of Heparin elution buffer)

 

  1. Recommended fraction collector setting: 250drops/fraction or 8ml/fraction

 

  1. Protein should start to be eluted at 1.2~1.4M NaCl

 

  1. Based on protein elution profile, take samples from fractions and run 15% SDS-PAGE decide which fraction should be pooled (see Fig.2)

 

  1. Pool the fractions

 

  1. Check the protein concentration at O.D280 (extinction coefficient : 1.26)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


Figure 2. Typical Heparin column elution profile and SDS-gel of wild-type FGF

 

 

Press Dialysis

 

: This step is for desalting (:Heparin elution pool contains high NaCl concentration) and concentrating (:compared to Ni-NTA elution, Heparin elution range is broad therefore protein is diluted in large volume) the protein. If next dialysis step will be done extensively, press dialysis can be skipped.

 

 

Crystal buffer (xtal buffer)

Buffer

Composition

Amount for 1L

pH

X-tal buffer

50mM NaPi

100mM NaCl

10mM (NH4)2SO4

2mM DTT

0.5mM EDTA

Dibasic (0.5M): 80.95ml and Monobasic (1M): 9.5ml

5.844gm

1.3214gm

0.3084gm

1ml of 0.5M stock

 

7.5

 

  1. Using amicon concentrator and 10kDa cutoff membrane, concentrate Heparin elution pool until ~1mg/ml concentration

 

  1. Add xtal buffer (2~3 times volume of protein) and concentrate till ~1mg/ml:

 

  1. Repeat [concentration adding xtal buffer] several times

 

Dialysis

 

  1. Using 6~8kDa cutoff dialysis membrane, do dialysis with xtal buffer: ~1/10000 dialysis should be enough

 

  1. Filter protein with 0.2 um filter unit

 

  1. Check the final protein concentration at O.D280

 

  1. Aliquot protein and store at -80 C

 

2007 Blaber lab