The expression and analysis of tetraspanin large extracellular domains

There are 33 tetraspanin proteins
in mammals and these are Widely expressed on the
majority of cells and tissues. Tetraspanin
proteins are differentiated from other
tetraspan
proteins by a number of conserved motifs,
in particular an absolutely conserved Cys-
Cys-Gly motif
in the large
extracellular
loop (EC2). This EC2 domain is
the most
variable region between
tetraspanin proteins
in length and amino acid sequence, and
consequently
is
the most
intriguing domain. Soluble
recombinant EC2 domains have
been used to study tetraspanin function,
and so one of the main aims of this work was
to
optimise
the production of GST-EC2
proteins
from CD9, CD81, CD63, CD151.
Methods were developed
to produce these domains in large quantifies, and they were
shown
to have biological
activity
in
the inhibition of giant cell
formation in human
monocytes. The soluble EC2 domains from CD63, CD9, CD81 and CD151 were all
shown
to potently
inhibit HIV-1 infection of macrophages.
Infection of
the two major
HIV-1 strains R5 and X4 was prevented by all the tetraspanins tested, suggesting an
involvement of a tetraspanin enriched microdomain. The inhibition was macrophage
specific since
infection of PBMC was not prevented.
'
Another aim was
to clone and
express EC2 domains from CD82 and CD231. CD82 and CD231 EC2 domains are
larger and consequently proved more difficult to express. Both were cloned successfully
into the pGEX-2T expression vector and CD82 was expressed
in Origami cells
(Novagen). CD82 EC2 was considered
to be a valuable tool to make because of the
involvement of CD82 in tumor cell metastasis. The original aim of
the work was
to
characterise the binding between tetraspanin EC2 domains and primary
ligands.
However, no appropriate
ligands were
found. We did
show by ELISA that mouse, but
not human, CD9 bound to murine PGS17 but we could not detect the reported binding
between CD9 and
fibronectin. Additionally, efforts were made to clone and express
two
immunoglobulin-like proteins
that had
recently been identified as CD9 and CD81
primary binding partners. The cloning of
full length EWI DNA as well and double Ig
domains was carried out successfully
into four different expression vectors, however, no
protein expression was'detected
in eukaryotic and a range of prokaryotic expression
systems. Structural studies were performed using CD63 EC2. Strategies to cleave GST
and to purify the EC2 domain were successfully developed. Instability problems of
CD63 EC2 in the absence of GST did not allow
for concentration of
the cleaved product
to levels
required
for crystal
trials. CD63 EC2-His6
recombinant protein was used
in X-
ray crystallography studies, and over 1000 crystallisation conditions were tested in
preliminary structural studies.