General Cloning Strategies for the Petm Series

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:

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-TEV-protease site-controlinsert-NcoI-TAATAA-rest of MCS.

Tags are : GST, MBP, dsb, dsbll, His-GST, His-MBP, CBC.

(remark: in preparation is a His-zzTag-version)

(note: GS recommends the addition of charged aa´s to the N- and C-terminus 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"95°C

5"58°C5 cycles

5"72°C

5"95°C

5"62-64°C15-20-25 cycles

5"72°C

15´72°C

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: 5´ ligation kit

Fermentas: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:20mMTris pH8.0

10mMImidazol

150mMNaCl

2mMß-ME

0.2%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

ElutionbufferLysisbuffer 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 purification12159-1651998

1534-391999

19312-3182000

Nature Biotech 17 july 1999, p691 Terwilliger et al.,