XXII. BIOCHEMICKÝ ZJAZD - Jesseniova lekárska fakulta

Posters
20.
EUKarYOTIC EXPrESSION as an INDISPENSaBLE TOOL for
prEParaTION of naTIve DIMErIC forMS of NK CELL C-TYPE LECTIN-
LIKE rECEPTOrs
Ondřej Vaněk 1 , Petra Celadová 1 , Jan Bláha 1 , Daniel Kavan 2 ,
Petr Pompach 2 and Karel Bezouška 1,2
1
Department of biochemistry PřF UK Praha, 2 Department of immunology
and gnotobiology MBÚ AVČR Praha
Natural killer cells are able to recognize and kill a variety of tumor and infected cells. The
recognition is mediated by wide repertoire of cell surface receptors,
both activation and inhibitory, belonging to two main structural families: immunoglobulinlike
and C-type lectin-like. While ligands for the Ig-like receptors were shown to be MHC
gp I proteins, ligands only for some of the lectin-like receptors are known up to date.
Both families share relatively weak binding characteristics and in the case of lectin-like
receptors, in vitro oligomerization clearly improves binding through increase in avidity.
However, these lectin-like receptors were described mostly as dimeric in vivo and this
minimal oligomer would be also the best for structural studies. Moreover, there is no
crystal structure known of extracellular domain of any of these receptors in its full-length
natural dimeric form; probably because prokaryotic expression of native dimers is rather
difficult. Here we present successful design, cloning and production of dimeric forms
of some mouse, rat and human NK cell lectin-like receptors belonging to NKRP1 and Clr
families. Full-length extracellular receptor parts were expressed via transient transfection
of HEK293 cell lines in suspension culture either alone or as a cleavable fusion with Fc
fragment of human IgG1 to promote native dimerization. This fusion strategy resulted in
cleaved pure covalent dimers of e.g. rat Clrb and purely dimeric Fc fusion of rat NKRP1B
proteins and their structural characterization is currently under way.
XXII. Biochemistry Congress, Martin
135