Engineered PQQ-Glucose Dehydrogenase as a Universal Biosensor Platform.
Journal of the American Chemical Society 138(32):10108 (2016)
Biosensors with direct electron output hold promise for nearly seamless integration with portable electronic devices. However, so far, they have been based on naturally occurring enzymes that significantly limit the spectrum of detectable analytes. Here, we present a novel biosensor architecture...
Performance benchmarking of four cell-free protein expression systems.
Biotechnology and Bioengineering 113(2):292 (2016)
Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in per...
Engineering PQQ-glucose dehydrogenase into an allosteric electrochemical Ca(2+) sensor.
Chemical Communications 52(3):485 (2016)
Electrochemical biosensors convert biological events to an electrical current. To date most electrochemical biosensors exploit activities of naturally occurring enzymes. Here we demonstrated that insertion of a calmodulin domain into the redox enzyme PQQ-glucose dehydrogenase resulted in a selec...
Mammalian farnesyltransferase α subunit regulates vacuolar protein sorting-associated protein 4A (Vps4A)--dependent intracellular trafficking through recycling endosomes.
Biochemical and Biophysical Research Communicat... 468(4):580 (2015)
The protein farnesyltransferase (FTase) mediates posttranslational modification of proteins with isoprenoid lipids. FTase is a heterodimer and although the β subunit harbors the active site, it requires the α subunit for its activity. Here we explore the other functions of the FTase α subunit in...
A highly sensitive prenylation assay reveals in vivo effects of bisphosphonate drug on the Rab prenylome of macrophages outside the skeleton.
Small GTPases 6(4):202 (2015)
Bisphosphonate drugs such as zoledronic acid (ZOL), used for the treatment of common bone disorders, target the skeleton and inhibit bone resorption by preventing the prenylation of small GTPases in bone-destroying osteoclasts. Increasing evidence indicates that bisphosphonates also have pleiotr...
Semisynthetic tRNA complement mediates in vitro protein synthesis.
Journal of the American Chemical Society 137(13):4404 (2015)
Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their util...
Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems.
Journal of Biotechnology 195:1 (2015)
Although numerous techniques for protein expression and production are available the pace of genome sequencing outstrips our ability to analyze the encoded proteins. To address this bottleneck, we have established a system for parallelized cloning, DNA production and cell-free expression of larg...
Synthetic protein switches: design principles and applications.
Trends in Biotechnology 33(2):101 (2015)
Protein switches are ubiquitous in biological signal transduction systems, enabling cells to sense and respond to a variety of molecular queues in a rapid, specific, and integrated fashion. Analogously, tailor-engineered protein switches with custom input and output functions have become invalua...
Towards the systematic mapping and engineering of the protein prenylation machinery in Saccharomyces cerevisiae.
PLoS ONE 10(3):e0120716 (2015)
Protein prenylation is a widespread and highly conserved eukaryotic post-translational modification that endows proteins with the ability to reversibly attach to intracellular membranes. The dynamic interaction of prenylated proteins with intracellular membranes is essential for their signalling...
Structural insights into the organization of the cavin membrane coat complex.
Developmental Cell 31(4):405 (2014)
Caveolae are cell-surface membrane invaginations that play critical roles in cellular processes including signaling and membrane homeostasis. The cavin proteins, in cooperation with caveolins, are essential for caveola formation. Here we show that a minimal N-terminal domain of the cavins, terme...
Protease-based synthetic sensing and signal amplification.
PNAS 111(45):15934 (2014)
The bottom-up design of protein-based signaling networks is a key goal of synthetic biology; yet, it remains elusive due to our inability to tailor-make signal transducers and receptors that can be readily compiled into defined signaling networks. Here, we report a generic approach for the const...
Increased Polyubiquitination and Proteasomal Degradation of a Munc18-1 Disease-Linked Mutant Causes Temperature-Sensitive Defect in Exocytosis
Cell Reports 9(1):206 (2014)
Munc18-1 is a critical component of the core machinery controlling neuroexocytosis. Recently, mutations in Munc18-1 leading to the development of early infantile epileptic encephalopathy have been discovered. However, which degradative pathway controls Munc18-1 levels and how it impact...
Rapid mapping of interactions between Human SNX-BAR proteins measured in vitro by AlphaScreen and single-molecule spectroscopy.
Molecular & Cellular Proteomics 13(9):2233 (2014)
Protein dimerization and oligomerization is commonly used by nature to increase the structural and functional complexity of proteins. Regulated protein assembly is essential to transfer information in signaling, transcriptional, and membrane trafficking events. Here we show that a combination of...
Cortactin scaffolds Arp2/3 and WAVE2 at the epithelial zonula adherens.
Journal of Biological Chemistry 289(11):7764 (2014)
Cadherin junctions arise from the integrated action of cell adhesion, signaling, and the cytoskeleton. At the zonula adherens (ZA), a WAVE2-Arp2/3 actin nucleation apparatus is necessary for junctional tension and integrity. But how this is coordinated with cadherin adhesion is not known. We now...
Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae.
eLife 3:e01434 (2014)
In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes...
Production of eukaryotic cell-free lysate from Leishmania tarentolae.
Methods in Molecular Biology 1118:1 (2014)
In this chapter, we describe the production and application of a eukaryotic cell-free expression system based on Leishmania tarentolae. This single-celled flagellate allows straightforward and inexpensive cultivation in flasks or bioreactors. Unlike many other Leishmania species, it is nonpathog...
Lipid-Anchored Ras is Sorted by Membrane Curvature Both In Vitro and in Living Cells
Biophysical Journal 104(2):96a (2013)
Rab GTPase prenylation hierarchy and its potential role in choroideremia disease.
PLoS ONE 8(12):e81758 (2013)
Protein prenylation is a widespread post-translational modification in eukaryotes that plays a crucial role in membrane targeting and signal transduction. RabGTPases is the largest group of post-translationally C-terminally geranylgeranylated. All Rabs are processed by Rab geranylgeranyl-transfe...
Subunit organisation of in vitro reconstituted HOPS and CORVET multisubunit membrane tethering complexes.
PLoS ONE 8(12):e81534 (2013)
Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protoz...
Cellular FRET-Biosensors to Detect Membrane Targeting Inhibitors of N-Myristoylated Proteins.
PLoS ONE 8(6):e66425 (2013)
Hundreds of eukaryotic signaling proteins require myristoylation to functionally associate with intracellular membranes. N-myristoyl transferases (NMT) responsible for this modification are established drug targets in cancer and infectious diseases. Here we describe NANOMS (NANOclustering and My...