-Purification of His-tagged wild-type FGF-

Expression (1L scale)

M9 media composition for IL

Na2HPO4 (Diabasic) : 12.0gm

KH2PO4 : 3.0gm

NaCl : 0.5gm

NH4Cl : 1gm

ddH2O : 987ml

à 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

à add these additives right before starting culture

  1. Inoculate single colony to 100ml of M9 media (w/ ampicillin: 0.15mg/ml media) à grow culture at 37°C, ~270rpm, for ~18hr
  1. Pour the overnight culture to 1L M9 media (w/ ampicillin: 0.15mg/ml media) à grow culture at 37°C 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 28°C à incubate for 5~6hr
  1. Harvest cells by centrifuging at 6,000 rpm for 15min
  1. Keep cell pellet at -20°C 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 à 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 à 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 à 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

7

Made by Jihun Lee/Blaber Lab

July 22, 2006