Table 2.
Protein | N-terminally taggeda |
C-terminally tagged |
||||||
---|---|---|---|---|---|---|---|---|
Solubilityb | Cleavedc | Crystals, diffraction limit | Solvedd | Solubilityb | Cleavedc (%) | Crystals, dif fraction limit | Solvedd | |
AsbA | 0.5 | + | Microcrystals | – | 0.5 | 10–20e | – | – |
AsbB | 3 | 100% | 4.5 Å | – | 3 | 80 | 2 Å | In progress |
AsbC | 2 | 50% | – | – | 3 | 80 | + no diffraction | – |
AsbD | 3 | 90% | – | – | 2 | 100 | – | – |
AsbE | 3 | 100% | + no diffraction | – | 0 | – | – | – |
AsbF | 3 | 0% | + no diffraction | – | 3 | 100 | 2 12 Å | 3dx5 |
RimM | 3 | 40% | Microcrystals | – | 1 | 100 | – | – |
CaiA | 3 | 0% | – | – | 3 | 100 | 3.1 Å | – |
DapE | 3 | 0% | – | – | 3 | 100 | 2.5 Å | 3ic1 |
Crl | 3 | 100% | 7.5 Å | – | 2 | 100 | + no diffraction | – |
RecF | 2 | 100% | 10 Å | – | 1 | 0 | – | – |
PmbA | 3 | 50% | 7.5 Å | – | 0.5 | n/a | – | – |
YwiE | 2f | – | – | – | 2 | 0 | 7Å | – |
BF1701 | 3f | – | – | – | 3 | 50 | 3.5 Å | – |
CD3330 | 2f | – | – | – | 3 | 100 | 2.02 Å | 3ivp |
TetR | 3 doubletg | 100% doubletg | 3.5 Å | – | 3 | 0 | 2.96 Å | 3f0c |
Expression from vector pMCSG7 or pMCSG19 gave N-terminal tags with His6-TEV site or MBP-TVMV site—His6-TEV site, respectively
Solubility scores were based on yields of soluble protein on gels, where 0 indicates no soluble protein, and 0.5 means very low; 1—low; 2—moderate; and 3—very good production of soluble protein
Cleavage with TEV protease is given in approximate percentage (cleavage estimated by visual evaluation of gels)
Protein Data Bank ID numbers are given for deposited structures
Since cleavage was very poor, only uncut protein was used in crystallization experiments
Poor or no Ni-binding of the protein at the purification stage
Purification yielded a doublet of proteins of similar molecular weight, both of which cleaved with TEV protease