Name: Petra Franzén (UU)
Position: Laboratory Engineer, Expression Laboratory
Funding ends: 31 December, 2000
Name: Eva Davey (UU)
Position: Laboratory Assistant, Expression Laboratory
Funding ends: 31 December, 2001
We have started a pilot project to study proteins from the Trypanosoma cruzi parasite using a "structural genomics" approach. Dr. Torsten Unge heads the expression studies. Three cosmid vectors (provided by Dr. Björn Andersson, Medical Genetics, Uppsala University) containing genes from T. cruzi were initially randomly chosen for the preliminary experiments. Seven genes were selected based on the likelihood of a successful expression in E. coli. The sizes of the gene products were in the range of 35-45 kDa and they contained few cysteine residues. Six of the genes could be directly isolated by PCR without having to redesign the primers or run nested PCR. Expression in E. coli was successful for 5 of the proteins. Modifications of the expression conditions resulted in soluble material for at least 4 of them. Work is now in progress with large scale production and crystallisation. During these preliminary experiments a cloning strategy was designed, which involves design of the initial PCR primers in such a way that expression can be done in bacteria with or without thioredoxin fused to the N-terminus. Secondly, the peptide sequences are provided with a N-terminal histidine tail for affinity purification. Finally, the genes can conveniently be transferred to a yeast expression system (Pichia pastoris) through restriction sites which were introduced in the initial PCR.
All members of the structural community in Uppsala use the equipment park. For example, the group of Prof. Sherry Mowbray used the Äkta for purification in her PDGF, phosphatase and ribokinase projects. The fermenter, shakers, centrifuge and other equipment for bacterial growth and preps were used for these projects as well. She reports that it has been most useful to have things centralised like this, and it is much more efficient than a system spread among different labs.
Other projects making use of the laboratory include:
* STRUCTURE AND FUNCTION OF A MOLECULAR THERMOMETER:
STUDIES ON TLPA, THE REGULATOR PROTEIN OF BACTERIAL
VIRULENCE
(Dr. Karin Valegård, Dept.Biochemistry, Uppsala
University)
The main goal of the project is to develop our understanding
of the structure and the mechanism of the thermosensing
gene regulator protein, TlpA, from the pathogenic bacterium
Salmonella typhimurium. Temperature is an important
factor affecting biochemical and physiological processes
in living systems, and a classical inducer of virulence
in pathogenic bacteria. Very little is known on how
cells sense temperature and concomitantly adjust gene
expression. TlpA has the capacity to both sense environmental
temperature, and to adjust repression of gene expression
as a response to temperature changes. We used facilities
in the expression lab to purify TlpA. Small crystals
have already been obtained which show diffraction.
* STRUCTURAL STUDIES ON MONONUCLEAR FERROUS ENZYMES
IN THE PENICILLIN AND CEPHALOSPORIN BIOSYNTHETIC PATHWAY
(Dr. Karin Valegård, Prof. Janos Hajdu, Dept.Biochemistry,
Uppsala University)
Despite the fact that beta-lactams have been known for
over 50 years there is still no efficient synthesis
of these antibiotics. Isopenicillin N synthase (IPNS)
utilises iron and molecular oxygen to remove four hydrogen
atoms from a linear tripeptide precursor of penicillins,
and thereby synthesises the labile and strained ring
structure of these antibiotics in a single event. There
is no synthetic precedent for this unique process.
An understanding of the reaction catalysed by IPNS
and the related ring expansion enzyme, deacetoxycephalosporin
C synthetase (REX or DAOCS, that catalyses the formation
of cephalosporins from penicillins), may lead to the
invention of new chemical reactions with application
in medicine, science, and industry. The structure of
both of these enzymes have now been solved and numerous
complexes produced. The expression lab played a vital
role in these projects and allowed us to keep an edge
on our competitors. These studies would not have been
done by us without these facilities.
* THE ISOLATED C-DOMAIN OF CYTOCHROME CD1 NITRITE REDUCTASE/OXIDASE
(Dr. Elles Steensma, Miss Tove Sjögren and Prof.
Janos Hajdu, Dept. Biochemistry, Uppsala University)
We have used the SBNet expression lab for all microbiology
and molecular biology work on the isolated c-domain
of cytochrome cd1 nitrite reductase/oxidase. Dr. Steensma
has received advice about several molecular biological
techniques and help with protocols. Furthermore, she
used the (biochemical) equipment such as the autoclave,
centrifuges and the PHAST and BIORAD systems. Thanks
to the equipment and the advice she was able to purify
the protein (containing the right amino acid sequence)
in relatively large quantities and solve the structure
by NMR. Therefore, the SBNet expression lab has been
instrumental for the success of this project. Miss
Sjögren has used the centrifuges, the autoclave
and the incubator for all X-ray structural studies
on the holoenzyme and its various mutants.
* ATTACHMENT OF ASYMMETRIC MACROMOLECULES TO SPECIFIC
BINDING SITES ON THE SURFACE OF ICOSAHEDRAL VIRUS CONSTRUCTS
(Dr Martin Svenda and Prof. Janos Hajdu, Dept.Biochemistry,
Uppsala University)
The study of low molecular weight proteins by electron
cryo-microscopy is problematic due to low S/N ratio.
One way of overcoming this problem is to attach such
low molecular weight proteins to regularly arranged
binding sites on a larger macromolecule. Thus a twofold
advantage is gained the larger macromolecule makes
it easier to locate and numerically align the protein
complex, and the symmetric binding gives an additional
improvement in the S/N ratio.
A generic method of attaching low molecular weight proteins
to sites related by icosahedral (60-fold) symmetry
on the surface of the tomato bushy stunt virus (TBSV)
has been developed by us in the expression lab. We
intend to use these constructs to obtains structures
for "uncrystallisable" proteins using single-particle
cryo-EM. We aim to employ this method to the study
of the structure of membrane proteins. We have cloned
the TBSV genome, made a number of different mutants
of the virus, expressed, purified and analysed these
mutants. We have also cloned, expressed and purified
several different proteins for use in the TBSV project.
All of the above mentioned work has been performed
in the expression lab. Without the existence of this
expression lab, much of the work would have been difficult
or impossible to perform.
* STRUCTURE OF RUBISCO FROM CHLAMYDOMONAS REINHARDTII
(Dr. Tom Taylor and Prof. Inger Andersson, Department
of Molecular Biology, SLU)
Wild type cells of the single-celled eukaryotic alga
Chlamydomonas reinhardtii have been grown and harvested.
From these cells the carbon-fixing enzyme Rubisco has
been purified. In addition to the wild type, several
mutant strains of Chlamydomonas expressing single and
double point mutations have been grown and harvested.
The structure has been solved.
Latest update at 8 March, 2001.