General Cloning strategies for the pETM series
by Gunter Stier and Arie Geerlof (EMBL-Heidelberg)
(Taken from a Talk by G. Stier at Como 2001)
They also have a website with a lot of helpful information available:
http://www.emblheidelberg.de/ExternalInfo/geerlof/draft_frames/index.html
check their protocol database: its really good!!!
Vectors:
general recommodations:
- Kanamycin resistance is better
thanAmpicillin (If you dont know why
check the appropriate textbooks!)
- use standard vectors with NcoI at 5´site
and usually KpnI, EcoRI or NotI at the 3´end.
- GS cloned a series of vectors using the
pET9d or pET24 backbone.
the general order is Tag1-Tag2-TEVprotease site-controlinsert-NcoITAATAA-rest of MCS.
Tags are : GST, MBP, dsb, dsbll, HisGST, His-MBP, CBC.
(remark: in preparation is a His-zzTagversion)
(note: GS recommends the addition of charged aa´s to the N- and Cterminus of your proteins!
note: there ius a plasmid available encodijng for LysS and the rare codon
tRNA´s.)
double expression experiments:
modified pET15a available in the lab (see Achim for details!)
GS has a pACYC9d for double expression experiments with the pLysS
promoter p15A from pACYC177 cloned into it.
Cloning of new constructs (Gunters q´nd)
1.) Primers and pcr
Primers should have a melting temperature of above 58°C (best 62°C or
more) for that part coding for the polypeptide. Add at least a CC 5´of the
restriction site you want to introduce.
For the pcr itself use Taq or Taq-mixes and the following cycle scheme:
5"
5"
5"
95°C
58°C
72°C
5"
5"
5"
95°C
62-64°C
72°C
15´
72°C
5 cycles
15-20-25 cycles
reactionvolume: 20-30 µl, reduce amount of NTPs (normal 100 pM each)
if problems.
note: no real kb limitation but use thionwall cups.
2.) Preparationof the vectors and inserts for ligation
- purify pcr-product via gel and gelelute.
- cut DNA 20-40 min and clean with quick spin nucleotide removal kit.
cut vector with first RE and dephosphorylate with SAP, purify and
cut vector with second RE and dephosphorylate with SAP gelpurify and
analyze both steps on gel
3.) ligation and transformations
Roche:
Fermentas:
5´ ligation kit
PEG Ligation kit (used by GS)
Trafo according to DMSO method (Inoue et al Gene, 1990; chemical
competent cells ~ 10 exp9 transformation efficiency):
10 min on ice
1 min 42°C plate on warm plates
note: plate 20 - 100µl on warm plates, do a vector control (usually no
positives!)
4. Expression tests: According to GS it is important to test different
colonies of the same Cell line for expression
Test 10-20 colonies. Pick them from the plate in 0,2ml LB in Eppis in the
presence of IPTG for 2 h.
harvest the cells and gain pellet.
sonicate in PBs in 0.5 - 1ml for 30 sec, use long tip.
Spin down, use supernatant for binding experiment
after incubation (30-60 min), wash beads once and run gel with the
samples obtained (sup, pellet, beads and supernatant of beads)
5. Purification and sequencing.
a.) sequencing
use T7 primer sequences from Novagen primers but extend for 5aa.
b.) purification
Lysis buffer:
20mM
10mM
150mM
2mM
0.2%
Tris pH8.0
Imidazol
NaCl
ß-ME
IGEPAL
(NP40 replacement from Sigma)
Lysozyme
DNase I
- 2-10 min on ice
- sonicate 3 x 30 sec
- spin 5-10 min 10000 xg
Test optimal amount of beads required to keep the binding sites saturated
20-30 min on roller, repeat the binding cycle with the sup again.
Wash cycles:
10 x CV Lysisbuffer +NP40
10 x CV Lysisbuffer
5 x CV Lysisbuffer + 1M NaCl
5 x CV Lysisbuffer + 40 mM Imidazol
Elutionbuffer
Lysisbuffer and 500 mM Imidazol
do column elution.
Change buffer using NAP 5 or 10 or PD10 columns
Amounts of beads to use for a start:
1ml beads per 5 ml supernatant
Literature:
David Waugh
NAR2000 28 # 50 e14
PNAS19976 94 8168-8172
Proteinexpression and purification
12
159-165
15
34-39
19
312-318
Nature Biotech 17 july 1999, p691 Terwilliger et al.,
1998
1999
2000