Biology Reference
In-Depth Information
(10 % DGCR8 buffer
B
). The fractions containing heme-
bound NC1 have a yellowish-brown color. Analyze the purity
of the fractions using sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE).
5. If the peak fractions are not >90 % pure, repeat the ion-
exchange chromatography step. Pool the fractions containing
relatively pure NC1. Dilute it 1:1 (v:v) with DGCR8 buffer
A
to reduce the salt concentration. Repeat
step 4
. The protein
may be stored at 4 °C overnight if desired.
6. Equilibrate the Superdex 200 column in SEC buffer (roughly
50 mL). Concentrate the fractions from the last ion-exchange
chromatography step down to ~550 μL using a centrifugal
concentrator. Filter the concentrated NC1 solution through a
membrane with 0.2 μm pores, and load the filtrate onto the
column. Collect 0.5 mL fractions. Heme-bound NC1 dimer
elutes at around 12.5 mL (
see
Note 5
).
7. Determine the NC1 protein concentration using UV-visible
absorption spectroscopy. Blank the spectrophotometer with
SEC buffer. Scan between 240 and 700 nm. An absorption
peak at 280 nm indicates that bacterial nucleic acids have been
successfully removed from the protein. NC1 dimer concentra-
tion =
A
280 nm
/
ε
280 nm
. Based on the amino acid sequence,
ε
280 nm
is estimated to be 94.5 mM
−1
cm
−1
(
ε
280 nm,apo
) [
35
]. This value
has been used in all our previous publications. However, our
recent unpublished measurements using microBCA assay indi-
cate
ε
280 nm
≈ 130 mM
−1
cm
−1
(
see
Note 6
).
8. Calculate the
A
450 nm
/
A
280 nm
ratio. If majority (>60 %) of the
NC1 protein is occupied by heme,
A
450 nm
/
A
280 nm
should be
between 0.40 and 0.53 (the higher the better). NC1 prepara-
tions with lower
A
450 nm
/
A
280 nm
ratios are less active (
see
Notes
7
and
8
).
9. The Fe(III) heme-bound NC1 protein may be stored at 4 °C,
with protection from light. Because this protein gradually loses
pri-miRNA processing activity for reasons not well under-
stood, we typically use it within a couple of days from the com-
pletion of purification.
3.3 Expression
and Purification
of Recombinant
Homo Sapiens
His
6
-Drosha
390-1374
1. His
6
-Drosha
390-1374
is expressed in Sf9 insect cells using a bacu-
lovirus system, following Invitrogen's standard protocols. The
cell pellets are stored in −80 °C freezer until purification.
2. Resuspend a pellet from 50 mL of insect cell culture in 30 mL
of ice-cold Drosha lysis buffer. Sonicate at 50 % power, 1-s on
and 1-s off, for a total of 4 min. To avoid overheating the lysate,
a 30-s break is taken after each minute of sonication and the
container is kept on ice throughout the sonication procedure.
3. Centrifuge the lysate at 45,000 ×
g
for 30 min at 4 °C.
