Biology Reference
In-Depth Information
and
EcoRI
sites of pET-24a(+) (kanamycin resistant) or pET-
17b (ampicillin resistant) vector. The PCR primers used in
cloning are CAGC
CATATG
GATGGAGAGACAAGTGTGC
(forward, the
NdeI
site underlined) and GCTC
GAATTC
AC
TTTCGAGTCTCCTCCCT (reverse, the
EcoRI
site underlined).
2.
E. coli
strain BL21-CodonPlus (DE3)-RIPL (Agilent
Technologies).
3. LB-Miller medium.
4. UV-visible absorption spectrophotometer equipped with a tur-
bidity cuvette holder.
5. Isopropyl β-
d
-1-thiogalactopyranoside (IPTG).
6. δ-aminolevulinic acid (δ-ALA).
7. High-speed centrifuge.
8. Sonics Vibra-Cell VCX 750 ultrasonic processor equipped
with a standard probe.
9. Chromatography systems such as ÄKTA Purifier and ÄKTA
Prime.
10. 5-mL HiTrap SP HP cation exchange column (GE Healthcare).
11. Superdex 200 10/300 GL gel filtration column (GE
Healthcare).
12. DGCR8 lysis buffer: 20 mM Tris-HCl pH 8.0, 100 mM NaCl,
1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithioth-
reitol (DTT) (
see
Note 1
).
13. DGCR8 buffer
A
: 20 mM Tris-HCl pH 8.0, 100 mM NaCl,
1 mM DTT.
14. DGCR8 buffer
B
: 20 mM Tris-HCl pH 8.0, 2 M NaCl, 1 mM
DTT.
15. SEC buffer: 20 mM Tris-HCl pH 8.0, 400 mM NaCl, 1 mM
DTT.
16. Centrifugal concentrator with a molecular weight cutoff of
30 kDa.
1. The His
6
-Drosha
390-1374
expression plasmid has the coding
sequence of amino acid residues 390-1,374 of human Drosha
(NCBI accession no. of full-length cDNA: NM_013235)
inserted between
BamHI
and
NotI
sites of pFastBac-HTb vector.
The PCR primers used in cloning are CGC
GGATCC
AAAGAGCCCGAGGAGACC (forward, the
BamHI
site
underlined) and GAGGATTAGA
GCGGCCGC
TTATTTCT
TGATGTCTTCAGTCTC (reverse, the
NotI
site underlined).
The recombinant His
6
-Drosha
390-1374
contains a His
6
-tag and a
TEV cleavage site at its N-terminus (
see
Note 2
).
2. Sf9 insect cells, culture medium, and transfection reagent.
3. Equipment same as the ones described for DGCR8 purification.
2.2 Expression and
Purification of Drosha
