Cell-free systems for the generation and characterization of membrane proteins, glycoproteins and novel antibody formats
Membrane proteins, glycoproteins and novel antibody formats have
become an important focus in the current efforts in structural and
functional analysis. Cell-free protein expression systems, in
particular those of eukaryotic origin, have recently been developed
as promising tools for the rapid and efficient production of a wide
variety of proteins. A huge number of these proteins, however,
require posttranslational modifications for optimum function. Several
membrane proteins have been expressed in vivo to date, most of
them being functionally, antigenically, and immunogenically similar
to their authentic counterparts. This is mainly due to the properties
of cultured eukaryotic cells, which carry out many types of
posttranslational modifications such as the addition of N- and Olinked
oligosaccharides, but also palmitoylation, myristylation, and
phosphorylation. Based on these versatile properties of cultured cell
lines, we have developed a technique for the standardized
production of translationally active eukaryotic lysates from insect
cells and CHO cells. In contrast to other cell-free protein synthesis
systems our homogenization procedure avoids any serious
breakdown of membrane vesicles already existing in the cytoplasm
of the prepared eukaryotic cells. We have demonstrated the
functional integrity of these subcellular components by showing
signal peptide cleavage as well as glycosylation in cell-free
systems. Moreover, we have expanded our cell-free protein
synthesis system by the insertion of orthogonal tRNA/synthetase
pairs to facilitate the cotranslational and site directed incorporation
of non-canonical building blocks. These fluorescently labeled and
chemoselective moieties enable the site-specific modification of de
novo synthesized antibodies, membrane proteins and
glycoproteins.
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