Publications by Kai Johnsson | EPFL Graph Search

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A stepwise approach to the development of artificial O6-alkylguanine-DNA alkyltransferases (AGTs) and its application to the in vitro selection of antibodies with highly reactive cysteine residues are described. As a result of low reactivity of O6-alkylated guanine as a substrate in SN2 reactions, a stepwise selection of artificial AGTs in which sulfuhydryl-specific reagents of the types II and III are first used to generate antibodies with reactive cysteine residues is ideal. These antibodies are then the

The invention relates to AGT mutants showing, when compared to the wild type human AGT, two or more advantageous properties selected from (a) reduced DNA interaction (b) localisation of the expressed protein in eukaryotic cells that is no longer restricted ...

The possibility to design proteins whose activities can be switched on and off by unrelated effector molecules would enable applications in various research areas, ranging from biosensing to synthetic biology. We describe here a general method to modulate the activity of a protein in response to the concentration of a specific effector. The approach is based on synthetic ligands that possess two mutually exclusive binding sites, one for the protein of interest and one for the effector. Tethering such a liga

The inter- and intramol. interactions between the different domains of the catalase-peroxidase KatG from Mycobacterium tuberculosis were analyzed using the two-hybrid assay. It was shown that the dimerization of the enzyme is due to a strong interaction of the first 99 amino acids of the N-terminal domain whereas the C-terminal domain does not play a role in the dimerization. In addn., an intramol. interaction between the N- and C-terminal domains was detected which might play a functional role in the mecha

We introduce color-shifting fluorophores that reversibly switch between a green and red fluorescent form through intramolecular spirocyclization. The equilibrium of the spirocyclization is environmentally sensitive and can be directly measured by determini ...

The authors describe a strategy for the synthesis of 'nanocrystal mols.', in which discrete nos. of Au nanocrystals are organized into spatially defined structures based on Watson-Crick base-pairing interactions. The authors attach single-stranded DNA oligonucleotides of defined length and sequence to individual nanocrystals, and these assemble into dimers and trimers on addn. of a complementary single-stranded DNA template. The authors anticipate that this approach should allow the construction of more com

We introduce here a new class of BODIPY-based Ca2+ indicators which can be derivatized with biological ligands that permit the localization of the indicators in living cells. The underivatized BODIPY-based Ca2+ indicator (BOCA-1) shows a 250-fold increase in fluorescence intensity upon Ca2+ binding. We also prepared its O6-benzylguanine (BG) derivative, BOCA-1-BG, which can be covalently and specifically linked to SNAP-tag fusion proteins in living cells. The indicator retains its properties as a highly sen

The quality and application of super-resolution fluorescence imaging greatly lie in the dyes' properties, including photostability, brightness, and Stokes shift. Here we report a synergistic strategy to simultaneously improve such properties of regular flu ...

We present a method for the specific labelling of fusion proteins with synthetic fluorophores in Zebrafish. The method uses the SNAP-tag technology and O-6-benzylguanine derivatives of various synthetic fluorophores. We demonstrate how the method can be used to label subcellular structures in Zebrafish such as the nucleus, cell membranes, and endosomal membranes. The stability of the synthetic fluorophores makes them attractive choices for long-term imaging and allows, unlike most of the autofluorescent pro

Synthetic bioactive hydrogels have been widely recognized as key elements of emerging strategies to engineer tissues. However, the current shortage of highly specific and biocompatible methods to form and functionalize these materials hampers their wide pharmaceutical and medical use. In particular, enzymatic reactions are underexplored for the synthesis of bioactive hydrogels. Here, we present an approach by which phosphopantetheinyl transferase (PPTase), a small (16.2 kDa) enzyme that plays a key role in

Cytochrome c peroxidase (I) from Saccharomyces cerevisiae was subjected to directed mol. evolution to generate mutants with increased activity against ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)]. Using a combination of DNA shuffling and satn. mutagenesis, mutants were isolated which possessed >20-fold increased activity against ABTS and a 70-fold increased specificity toward ABTS compared to the natural substrate. In contrast, activities against another small org. mol., guaiacol, were not

Phenylketonuria (PKU) is a rare metabolic disorder characterized by elevated phenylalanine (PHE) levels, requiring lifelong dietary management and regular monitoring. Current PHE monitoring methods, such as tandem mass spectrometry (FIA-MS/MS), are laboratory-dependent, necessitating sample transport and causing delays in result delivery. This emphasizes the need for point-of-care (PoC) solutions. We developed a bioluminescence-based PHE sensor integrated with the phenyx smartphone app for real-time monitor

Cyclic guanosine monophosphate (cGMP) is a ubiquitous second messenger, that regulates the cardiovascular system, brain functions and is involved in many pathological processes. To visualize cellular dynamics of cGMP levels, fluorescent protein-based sensors have been developed. However, these fluorescent sensors are not well suited to monitor changes in cGMP levels in large cell populations or in vivo. Here, we introduce a molecular recorder to transform transient changes in cGMP levels into permanent fluo

While gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter, suitable tools to measure its concentration in living cells with high spatiotemporal resolution are missing. Herein, we describe the first ratiometric fluorescent sensor for GABA ...

CLIP-tag is a self-labeling protein tag used for the specific fluorescence labeling of proteins. However, its low labeling speed and the poor cell permeability of its substrates result in low labeling efficiencies in live-cell applications. Here, we introduce a substrate optimized for live-cell applications, as well as an engineered CLIP-tag variant, CLIP-tag2, which reacts with the new substrate almost 1000-fold faster than the original CLIP-tag-substrate pair. CLIP-tag2 fusion proteins can be specifically

Split-protein sensors have become an important tool for the anal. of protein-protein interactions in living cells. We present here a combinatorial method for the generation of new split-protein sensors and demonstrate its application toward the (b/a)8-barrel enzyme N-(5'-phosphoribosyl)-anthranilate isomerase Trp1p from Saccharomyces cerevisiae. The generated split-Trp protein sensors allow for the detection of protein-protein interactions in the cytosol as well as the membrane by enabling trp1 cells to gro

A method for labeling acyl carrier protein (ACP) fusion proteins with a wide variety of different labels is disclosed. The method relies on the transfer of a label from a coenzyme A type substrate to an ACP fusion protein using a holo-acyl carrier protein synthase (ACPS) or a homologue thereof. The method allows detecting and manipulating the fusion protein, both in vitro and in vivo, by attaching molecules to the fusion proteins that introduce a new physical or chemical property to the fusion protein. Exam

While antibodies currently play a dominant role as affinity reagents in biological research and for diagnostics, a broad range of recombinant proteins are emerging as promising alternative affinity reagents in detection assays and quantification. DNA-media ...

Tryptophan metabolites in the kynurenine pathway are up-regulated by pro-inflammatory cytokines or glucocorticoids, and are linked to anti-inflammatory and immunosuppressive activities. In addition, they are up-regulated in pathologies such as cancer, auto ...

Kai Johnsson

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Towards the generation of artificial O6-alkylguanine-DNA alkyltransferases: In vitro selection of antibodies with reactive cysteine residues

Mutants of o6-alkylguanine-dna alkyltransferase

Modulating protein activity using tethered ligands with mutually exclusive binding sites

Inter- and intramolecular domain interactions of the catalase-peroxidase KatG from M. tuberculosis

Environmentally Sensitive Color-Shifting Fluorophores for Bioimaging

Organization of 'nanocrystal molecules' using DNA

Localizable and highly sensitive calcium indicator based on a BODIPY fluorophore

A synergistic strategy to develop photostable and bright dyes with long Stokes shift for nanoscopy

Labelling cell structures and tracking cell lineage in zebrafish using SNAP-tag

Phosphopantetheinyl transferase-catalyzed formation of bioactive hydrogels for tissue engineering

Changing the substrate specificity of cytochrome c peroxidase using directed evolution

Real-life application of a point-of-care biosensor for phenylalanine in patients with phenylketonuria

Development of a cGMP recorder with chemical labeling

A Fluorescent Sensor for GABA and Synthetic GABA(B) Receptor Ligands

Fast and Luminous: CLIP-tag2

Posttranslational covalent labeling reveals heterogeneous mobility of individual G protein-coupled receptors in living cells

Transforming a (beta/alpha)8-Barrel Enzyme into a Split-Protein Sensor through Directed Evolution

Methods for protein labeling based on acyl carrier protein

Protein tag-mediated conjugation of oligonucleotides to recombinant affinity binders for proximity ligation

Tetrahydrobiopterin Biosynthesis as a Potential Target of the Kynurenine Pathway Metabolite Xanthurenic Acid