Biomedical Engineering Reference
In-Depth Information
Fig. 3
Purifi cation of yeast eIF5 NIP1/TIP32 sub-complexes. Two related complexes of NIP1 (amino acids
249-806) and TIP32 (amino acids 198-595 or 20-595) were made by cloning StrepII-tagged NIP1 into
pOPINRSF (the vector was linearized with NcoI HindIII which removed the vector-derived N-terminal His-tag
and a C-terminally StrepII-tagged NIP1 was used as template to provide a StrepII-tagged protein) and the two
TIP32 variants into pOPINF (N-hexa-histidine). (
a
) Coomassie-stained polyacrylamide gel showing fractions of
a scaled-up purifi cation of the NIP1/TIF32 (amino acids 249-806 and either 198-595 or 20-595) complex.
These sub-complexes were purifi ed by Ni-NTA affi nity chromatography (lanes 1 and 2, respectively) and gel
fi ltration (lanes 3 and 4, respectively). (
b
) The western blots shown in (
c
) and (
d
) merged onto the gel in (
a
) to show
that a His-tagged and Strep-tagged protein have been purifi ed as a complex. (
c
) Anti-strep western blot (NIP-1)
against the gel in (
a
). (
d
) Anti-His western blot (TIF32) against the gel in (
a
)
suite of vectors using an inter-cistronic ribosome binding site/s as
the homology region (e.g., using pOPINF the 5
protein would
have N-terminal hexa-histidine tag, and the other protein/s are
untagged; [
36
]). Thus by combining multi-fragment In-Fusion™
and the family of pOPIN co-expression vectors larger complexes
may be assembled [
36
].
′
