Table 1.
Summary of 15N isotope-labeled Aβ42 purification from 1 L E. coli culture in auto induction minimal medium.a,b
| Purification steps | Volume (mL) | IFABP-Aβ42 fusion (mg) | Aβ-42 (mg) | % Recovery |
|---|---|---|---|---|
| Inclusion bodies dissolved in urea buffer (urea soluble portion) | 50 | ND | ND | ND |
| Ni–NTA column chromatography | 26 | 40 | 8.6c | 100d |
| Factor-Xa cleavage | 50 | 40 | 8.6c | ND |
| Overnight precipitation dissolved in urea buffer | 4 | – | 8.6c,e | ND |
| Mini Ni–NTA column chromatography | 4.5 | ND | ND | ND |
| HPLC (C18 reverse-phase) | 120 | ND | 6 | 70 |
a
3.8 g of wet cells harvested from 1 L culture.
b
Fusion protein (IFABP-Aβ42) represents about 32% of the expressed protein in bacterial lysate (soluble plus inclusion bodies) when estimated from the band intensity in ImageJ [26].
c
The amount of Aβ42 is calculated from mass ratio of Aβ42 and the IFABP-Aβ42 fusion protein.
d
The amount of fusion protein isolated from the affinity column was assumed 100% of the fusion protein in the bacterial lysate that can be isolated.
e
The precipitate and subsequent urea soluble component are expected to contain the full amount of Aβ-42 initially present. ND Not determined.