PSF
•What we do•
The Protein Structure Factory uses E. coli and yeast expression systems to generate human proteins in recombinant form for structural analyses. High throughput methods have been developed for cloning, characterisation of expression clones and large scale protein expression and purification.
Cloning strategy
Human cDNA fragments are cloned by
PCR, using cDNA clones supplied by the German
Resource Center as templates. We use restriction enzyme cloning exclusively.
All E. coli and yeast vectors have
been designed for accepting inserts with 5'-BamHI and 3'-NotI
overhangs.
PCR primers are equipped with tails including BamHI or NotI sites. If a cDNA fragment contains an internal BamHI or NotI site, alternative sites are used. This is done automatically by our primer design software. BamHI can be replaced by BglII, as the same overhangs are obtained upon cleavage with either enzyme. Alternatively, we use type II restriction enzymes, that cleave downstream of their recognition sequence and therefor can produce arbitrary overhangs. See the documentation of the DoveTail kit of Fermentas for details.
Specific 5' and 3'-primers are used for cloning into the pQStrep vectors and the corresponding yeast vectors. These vectors allow for expression of fusion proteins with N-terminal His-tag and C-terminal Strep tag II (see Figure). Vector primers are used in combination with specific 5'-primers, if PCR products are cloned into vectors that allow for expression of N-terminal tags only. These vector primers are M13-Forward or M13-Reverse, equipped with NotI site tails, since most of the cDNA templates we used are in vectors that contain M13 sequences.
We use the Expand kit of Roche or Pfx of Invitrogen for PCR. PCR primers were recently purchased of Metabion and Eurogentec. Primers are ordered in 96-well format. A pipetting robot is used for reformatting primers and cDNA templates.
Primers are designed to have a melting temperature of about 65°C. The PCR programme consists of 25 cycles of 30s 94°C, 30s 60°C, 2min 72°C, followed by 15 min final elongation at 72°C.
PCR is checked by agarose gel electrophoresis. Product size is recorded using the 1D-quantifier software of Phoretix. Successful PCRs are reformatted and purified on the pipetting robot, followed by restriction digest. Purification of PCR products and restriction fragments is performed with magnetic particles using a procedure developed in the group of Richard Reinhard at the Max Planck Institute of Moleculare Genetics.
DNA product yield is determined by UV-spectroscopy in a 96-well device (BioTek), and ligation is set up according to the measured concentrations. A molar ratio of 3:1 insert:vector and 5 ng vector is used. E. coli cells are transformed in 96-well format in a PCR machine by chemo-transformation/heat shock and are plated on standard round 82 mm agar plates. Four transformants are picked per transformation into 96-well microplates (filled with medium and antibiotics) and checked by colony PCR using vector primers. Two transformants with inserts are reformatted into microplates and subsequently subjected to protein expression analysis.
Expression clone characterisation
Deep 96-well microplates and SB medium, allowing for high density cell growth,
is used for small-scale protein expression. Protein purification via His-tag
or GST-tag is perfomed automatically on a Zinsser Speedy pipetting robot
under non-denaturing conditions. Whole cellular extracts and purification
eluates are subjected to SDS-PAGE followed by Coomassie staining (Figure).
We have published our automated protein purification
procedure here:
An automated method
for high-throughput protein purification applied to a comparison of His-tag
and GST-tag affinity chromatography.
© 2003 by V. Sievert, Konrad Büssow last changed 12 Sep 2006