Purification of scIHF

Purification of scIHF

1. grow cells in LB to OD600 0.4~ 0.6
2. Add 1 mM IPTG, induce for 4 h
3. Spin down cells
4. Resuspend in Lysis Buffer.

Insoluble fraction: use this buffer: 0.1 M NaH2PO4, 10 mM Tris, 0.1% NP-40, 8 M Urea, pH8.0

5. Sonicate
6. Spin and collect supernatant

Alternative steps: 7 to 11 for non tag protein

7. Precipitate with 50% - 80% ammonium sulfate.

8. Resuspend the pellet and dialyze against 100mM NaCl, 25mM HEPES pH 7.0 and 10% glycerol.

9. Load on a SP Sepharose column. 

10. Wash with 150mM and 250mM NaCl in 25mM HEPES pH 7.0

11. Elute with  350mM NaCl step in 25mM HEPES pH 7.0, 0.1mM EDTA, and 5% glycerol. 

7. Load to Ni column.
8. Wash with Lysis Buffer

9. Wash extensively with 50 mM NaH2PO4, 300 mM NaCl, 5 mM imidazole pH 8.0 to remove urea.

10. Elute with Elution Buffer 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0
11. Concentrate and then dialysis vs 0.1 M NaH2PO4, 0.1 mM EDTA, 1 mM DTT, 50 mM NaCl, 50% glycerol

12. Store at -80 oC.

Amino acid sequence of scIHF

beta
Nco1
cc atg gcg accaagtcagaattgatagaaagacttgccacccagcaatcgcacattcccgccaag
    M   A   T  K  S  E  L  I  E  R  L  A  T  Q  Q  S  H  I  P  A  K 
acggttgaagatgcagtaaaagagatgctggagcatatggcctcgactcttgcgcagggc
 T  V  E  D  A  V  K  E  M  L  E  H  M  A  S  T  L  A  Q  G 
 
 GGC AGC GGC GGT
  G   S   G   G
 
alpha
cttacaaaagctgaaatgtcagaatatctgtttgataagcttgggcttagcaag
 L  T  K  A  E  M  S  E  Y  L  F  D  K  L  G  L  S  K 
cgggatgccaaagaactggttgaactgtttttcgaagagatccgtcgcgctctggaaaac
 R  D  A  K  E  L  V  E  L  F  F  E  E  I  R  R  A  L  E  N 
ggcgaacaggtgaaactctctggttttggtaacttcgatctgcgtgataagaatcaacgc
 G  E  Q  V  K  L  S  G  F  G  N  F  D  L  R  D  K  N  Q  R 
ccgggacgtaacccgaaaacgggcgaggatattcccattacagcacggcgcgtggtgacc
 P  G  R  N  P  K  T  G  E  D  I  P  I  T  A  R  R  V  V  T 
ttcagacccgggcagaagttaaaaagccgggtcgaaaacgct
 F  R  P  G  Q  K  L  K  S  R  V  E  N  A 

GGC GGC GGT
 G   G   G
 
beta
gagcgtattgaaatccgcggtttcggcagtttctctttgcactaccgcgcaccacgtacc
 E  R  I  E  I  R  G  F  G  S  F  S  L  H  Y  R  A  P  R  T 
ggacgtaatccgaagactggcgataaagtagaactggaaggaaaatacgttcctcacttt
 G  R  N  P  K  T  G  D  K  V  E  L  E  G  K  Y  V  P  H  F 
aaacctggtaaagaactgcgcgatcgcgccaatatttacggt
 K  P  G  K  E  L  R  D  R  A  N  I  Y  G 
                                          Xho1
 GGT AGC GGC  CAC CAC CAC CAC CAC CAC TGA ctc gag GATCag
  G   S   G