Construction

Instability
AA MW pI
index
ERS1-1(2.0kb) 1-688(688) 78521.9 6.72 35.34
ERS1-2(2.1kb) 1-706(706) 80509.2 6.62 35.68
ERS2-1(1.5kb) 161-688(528) 60847.8 8.2 35.06
ERS2-2(1.6kb) 161-706(546) 62835.1 7.93 35.52
ERSdGST 194 – 709 (539) 62106.1 7.63 35.21
 Solubility test
18º 37º 18º 37º
C C C C
0.5mM 1mM 0.5mM 1mM IPTG 0.5mM 1mM 0.5mM 1mM IPTG
IPTG IPTG IPTG IPTG IPTG IPTG
M C T S P T S P T S P T S P C T S P MT S P T S P T S P
100
100
70
70

 ERS1-2 pET28a BL*  ERS1-1 pET28a BL*

Solubility test
18º 37º 18º 37º
C C C C
0.5mM 1mM 0.5mM 1mM IPTG 0.5mM 1mM 0.5mM 1mM IPTG
IPTG IPTG IPTG IPTG IPTG IPTG
C M T S P T S P T S P T S P C T S P T MS P T S P T S P

100 100
70
70

 ERS1-2 pET28a Ro1  ERS1-1 pET28a Ro1

Solubility test
18º 37º
18º 37º C C
0.5mM 1mM 0.5mM 1mM IPTG
0.5mM C 1mM 0.5mM C 1mM IPTG IPTG IPTG IPTG
IPTG IPTG IPTG C T S P T S P MT S P T S P
C T M S P T S P T S P T S P
100
100
70
70

 ERS1-2 pET28a Ro2  ERS1-1 pET28a Ro2

Cell culture and harvest of ERS1-2

 2L large culture

 O.D 600 : 0.615

 Induction : 1mM, incubated for 18hrs at 18ºC

 75mL of His-tag A buffer

 Sonication : 4 mins 30 secs (pulse on : 2 secs, off : 6 secs)

amplitude : 54%
 Affinity chromatography

M S Ft W 3 4 5 6 7 8 9 10 11 12 13 M 14 15 16 17 18 19 20 21 22 23

70 70

50 50

 His tag A buffer  His tag B buffer

 pH 8.0  pH 8.0
 20mM Tris  20mM Tris
 100mM NaCl  100mM NaCl
 5mM MgCl2  5mM MgCl2
 5mM β-  500mM Imidazole
Mercaptoethanol  5mM β-
Mercaptoethanol
 W : 2% of B buffer
 10 ~ 23
 Ni column
Ion exchange chromatography
M S Ft 1 2 3 4 5 6 7 8 9 10 1112 M 13 14 1516 17 1819 20 2122 2324 25 26

70 70
50
50

 Ion A buffer  Ion B buffer

 pH 8.0  pH 8.0
 20mM Tris  20mM Tris
 5mM DTT  1M NaCl
 5mM MgCl2
 5mM DTT

 8~10
 11~14
 15~18
 19~22

 Q column
 Gel filtration chromatography
M 1 3 5 7 9 11 13 15 17 19 21 232527 29 M 31 33 35 37 39 41 43 45 47 49 51 53 55

70 70

50 50

57 59 M 61 63 65 67 69 71 73 75 77 79 81 83 85 87 89 M 91 93 95 97 99 101 103 105 107 109 111

70 70

50
50
Gel filtration chromatography

 45 ~ 55, 71 ~ 81  Gel buffer

 pH 8.0
 ~120kDa  20mM Tris
 20mg/ml  100mM NaCl
 5mM MgCl2
 S200  1% Glycerol
 5mM DTT
Cell culture and harvest of ERS1-2

 2L large culture

 O.D 600 : 0.712

 Induction : 1mM, incubated for 18hrs at 18ºC

 75mL of His-tag A buffer

 Sonication : 5 mins (pulse on : 1 secs, off : 5 secs)

amplitude : 56%
 Affinity chromatography
M S Ft W 7 8 9 10 11 12 13 14 15 16 17

70

50

 His tag A buffer  His tag B buffer

 pH 8.0  pH 8.0
 20mM Tris  20mM Tris
 100mM NaCl  100mM NaCl
 5mM MgCl2  5mM MgCl2
 5mM β-  500mM Imidazole
Mercaptoethanol  5mM β-
Mercaptoethanol

 W : 3% of B buffer
 9 ~ 14
 Ni column
 Gel filtration chromatography

M 51 52 53 54 55 56 57 58 59 60 61 62 63 64 M 65 66 67 68 69 70 71 72 73 74 75 76 77 78

70 70

50 50

 Gel buffer
 pH 8.0
 20mM Tris
 100mM NaCl
 5mM MgCl2
 1% Glycerol
 5mM DTT
 59 ~ 68
 20mg/ml
 S200
Cell culture and harvest of ERS2-2, PRS and ERS

 2L large culture

 O.D 600 : 0.712

 Induction : 1mM, incubated for 18hrs at 18ºC

 75mL of His-tag A buffer

 Sonication : 5 mins (pulse on : 1 secs, off : 5 secs)

amplitude : 57%
 Affinity chromatography
M P S Ft W 6 7 8 9 10 11 12 13 14 15 P M S Ft W 5 6 7 8 9 10 11 12 13 14
70 70
50 50

Ro1 Ro2

 His tag A buffer  His tag B buffer

 pH 8.0  pH 8.0 Ro1
 20mM Tris  20mM Tris
 100mM NaCl  100mM NaCl
 5mM MgCl2  5mM MgCl2
 5mM β-ME  500mM Imidazole
 5mM β-ME

 W : 3% of B buffer
 Ni column Ro2
Gel filtration chromatography

M 66 67 68 69 70 71 72 73 74 75

70

50

 Gel buffer
 pH 8.0
 20mM Tris
 100mM NaCl
 5mM MgCl2
 1% Glycerol
 5mM DTT

 S200
Cell culture and harvest of ERS 1-2

 2L large culture

 O.D 600 : 0.630

 Induction : 1mM, incubated for 18hrs at 18ºC

 30mL of His-tag A buffer

 Sonication : 5 mins (pulse on : 1 secs, off : 5 secs)

amplitude : 56%
 Affinity chromatography

M S Ft W 9 10 11 12 1314 151617 1819

70

50

 His tag A buffer  His tag B buffer

 pH 8.0  pH 8.0
 20mM Tris  20mM Tris
 100mM NaCl  100mM NaCl
 5mM MgCl2  5mM MgCl2
 5mM β-ME  500mM Imidazole
 5mM β-ME

 W : 3% of B buffer
 10 ~ 19
 Ni column
Ion exchange chromatography

M 8 9 10 11 1213 141516 1718 19 20 M 21 22 23 24 25 2627 28293031 32 3334

70 70
50 50

 Ion A buffer  Ion B buffer

 pH 8.0  pH 8.0
 20mM Tris  20mM Tris
 5mM MgCl2  1M NaCl
 5mM DTT  5mM MgCl2
 5mM DTT

 8 ~ 13
 Resource Q
 Gel filtration chromatography

M 153 54 55 56 57 58 59 60 61 62 63 64 65 66 67 M 68 69 70 71 72 73 74 75 76 77 78 79

70 70

50 50

 Gel buffer
 pH 8.0
 20mM Tris
 100mM NaCl
 5mM MgCl2
 5mM DTT

 164 ~ 175
 S200
Cell culture and harvest of ERS2-2 pET28a Ro1

 6L large culture

 O.D 600 : 0.630

 Induction : 1mM, incubated for 18hrs at 18ºC

 90mL of His-tag A buffer

 Sonication : 5 mins (pulse on : 2 secs, off : 5 secs)

amplitude : 58%
 Affinity chromatography

M S Ft W 6 7 8 9 10 1112 13 14 1516

70
50

 His tag A buffer  His tag B buffer

 pH 8.0  pH 8.0
 20mM Tris  20mM Tris
 100mM NaCl  100mM NaCl
 5mM MgCl2  5mM MgCl2
 5mM β-ME  500mM Imidazole
 5mM β-ME

 W : 3% of B buffer
 7 ~ 15
 Ni column
 Gel filtration chromatography
M 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 M 79 80 81 82 83 84 85 86 87 88 89 90 91 92
70
70
50
50

 Gel buffer
 pH 8.0
 20mM Tris
 100mM NaCl
 5mM MgCl2
 5mM DTT

 64 ~ 77
 S200
 Monomer
Cell culture and harvest of ERS 1-2

 6L large culture

 O.D 600 : 0.630

 Induction : 1mM, incubated for 18hrs at 18ºC

 30mL of His-tag A buffer/2L

 Sonication : 5 mins (pulse on : 1 secs, off : 5 secs)

amplitude : 56%
 Affinity chromatography

M S W 13 1415 16 1718 19 20 212223 24

70

50

 His tag A buffer  His tag B buffer

 pH 8.0  pH 8.0
 20mM Tris  20mM Tris
 100mM NaCl  100mM NaCl
 5mM MgCl2  5mM MgCl2
 5mM β-ME  500mM Imidazole
 5mM β-ME

 W : 3% of B buffer
 15 ~ 22
 Ni column
 Gel filtration chromatography

M 43 44 45 46 47 48 49 50 5152 53 54 55 56 57 M 58 59 60 61 62 6364 65 666768 69

70
70
50 50

 Gel buffer
 pH 8.0
 20mM Tris
 100mM NaCl
 5mM MgCl2
 5mM DTT

 43 ~ 56 => Resource Q
 57 ~ 69 => for complex
 S200
Ion exchange chromatography

24 M 25 26 27 28 29 30 31 32 33 34 35 36
70

50

 Ion A buffer  Ion B buffer

 pH 8.0  pH 8.0
 20mM Tris  20mM Tris
 5mM MgCl2  1M NaCl
 5mM DTT  5mM MgCl2
 5mM DTT

 24 ~ 28
 Resource Q
Affinity chromatography

M Ft 11 12 13 1415 16 17 18 19 20 21 22 23  His tag B buffer

 His tag A buffer
 pH 8.0  pH 8.0
75  20mM Tris  20mM Tris
 100mM NaCl  100mM NaCl
 5mM MgCl2  5mM MgCl2
37  500mM Imidazole
 5mM β-ME
 5mM β-ME

 W : 3% of B buffer
 Ni column
 12 ~ 20
 Ion exchange chromatography

M Ft W 20 21 22 23 24 25 26 27 28 29 30 31
75

37

 Ion A buffer  Ion B buffer

 pH 8.0  pH 8.0
 20mM Tris  20mM Tris
 5mM DTT  1M NaCl
 5mM DTT

 23 - 27
 Q column
 Gel filtration chromatography

M 31 32 33 34 35 36 37 38 39 40 41 42 43 44

75

37

 Final buffer
 pH 8.0
 20mM Tris
 100mM NaCl
 5mM MgCl2
 5mM DTT

 34 ~ 37
 S200(120ml)
 Gel filtration chromatography
M 31 32 33 34 35 36 37 38 39 40 41 42 43 44

75
ERS_706

37
AIMP2_34S
25
MRS_205
20
AIMP3_Full

 Final buffer
 pH 8.0
 20mM Tris
 100mM NaCl
 5mM MgCl2
 5mM DTT

 33 ~ 37
 S200(120ml)
Cell culture and harvest of ERSdGST pET21a Ro1

 2L large culture

 O.D 600 : 0.590

 Induction : 1mM, incubated for 18hrs at 18ºC

 50mL of His-tag A buffer

 Sonication : 5 mins (pulse on : 1 secs, off : 5 secs)

amplitude : 58%
Affinity chromatography

M S Ft W 10 11 12 13 14 15 16 17 18 19 20
 His tag A buffer  His tag B buffer
 pH 7.5  pH 7.5
75  20mM Tris  20mM Tris
 100mM NaCl  100mM NaCl
50  5mM MgCl2  5mM MgCl2
 5mM b-ME  500mM Imidazole
 5mM b-ME

 W : 3% of B buffer
 Ni column
 16 ~ 22
 Gel filtration chromatography

M 41 42 43 44 45 46 48 51 52 53 54 55

75

50

 Final buffer
 pH 7.5
 20mM Tris
 100mM NaCl
 5mM MgCl2
 5mM DTT
 51 ~ 55
 S200(120ml)
 20 mg/ml
LegAS4 – 8mer peptide complex

 Precipitant : Jeffamine ED-2001

 0.1M Imidazole/HCl
 pH 7.0