2 research outputs found
A Modular Toolkit for Generating <i>Pichia pastoris</i> Secretion Libraries
Yeasts
are powerful eukaryotic hosts for the production of recombinant
proteins due to their rapid growth to high cell densities and ease
of genetic modification. For large-scale industrial production, secretion
of a protein offers the advantage of simple and efficient downstream
purification that avoids costly cell rupture, denaturation and refolding.
The methylotrophic yeast <i>Pichia pastoris</i> (<i>Komagataella phaffi</i>) is a well-established expression host
that has the ability to perform post-translational modifications and
is generally regarded as safe (GRAS). Nevertheless, optimization of
protein secretion in this host remains a challenge due to the multiple
steps involved during secretion and a lack of genetic tools to tune
this process. Here, we developed a toolkit of standardized regulatory
elements specific for <i>Pichia pastoris</i> allowing the
tuning of gene expression and choice of protein secretion tag. As
protein secretion is a complex process, these parts are compatible
with a hierarchical assembly method to enable the generation of large
and diverse secretion libraries in order to explore a wide range of
secretion constructs, achieve successful secretion, and better understand
the regulatory factors of importance to specific proteins of interest.
To assess the performance of these parts, we built and characterized
the expression and secretion efficiency of 124 constructs that combined
different regulatory elements with two fluorescent reporter proteins
(RFP, yEGFP). Intracellular expression from our promoters was comparatively
independent of whether RFP or yEGFP, and whether plasmid-based expression
or genomically integrated expression, was used. In contrast, secretion
efficiency significantly varied for different genes expressed using
identical regulatory elements, with differences in secretion efficiency
of >10-fold observed. These results highlight the importance of
generating
diverse secretion libraries when searching for optimal expression
conditions, and demonstrate that our toolkit is a valuable asset for
the creation of efficient microbial cell factories
6āAlkoxy-5-aryl-3-pyridinecarboxamides, a New Series of Bioavailable Cannabinoid Receptor Type 1 (CB1) Antagonists Including Peripherally Selective Compounds
We identified 6-alkoxy-5-aryl-3-pyridinecarboxamides
as potent
CB1 receptor antagonists with high selectivity over CB2 receptors.
The series was optimized to reduce lipophilicity compared to rimonabant
to achieve peripherally active molecules with minimal central effects.
Several compounds that showed high plasma exposures in rats were evaluated
in vivo to probe the contribution of central vs peripheral CB1 agonism
to metabolic improvement. Both rimonabant and <b>14g</b>, a
potent brain penetrant CB1 receptor antagonist, significantly reduced
the rate of body weight gain. However, <b>14h</b>, a molecule
with markedly reduced brain exposure, had no significant effect on
body weight. PK studies confirmed similarly high exposure of both <b>14h</b> and <b>14g</b> in the periphery but 10-fold lower
exposure in the brain for <b>14h</b>. On the basis of these
data, which are consistent with reported effects in tissue-specific
CB1 receptor KO mice, we conclude that the metabolic benefits of CB1
receptor antagonists are primarily centrally mediated as originally
believed