Kristian M. Müller

Bielefeld University · Faculty of Technology

Research on adeno-associated virus (AAV) and its recombinant vectors as well as on fluorescence microscopy imaging is rapidly progressing driven by clinical applications and new technologies, respectively. The topics converge, since high and super-resolution microscopes facilitate the study of spatial and temporal aspects of cellular virus biology....

Virus-like particles (VLPs) have been used for numerous pharmaceutical applications, particularly vaccination and drug delivery. Recombinant adeno-associated virus (rAAV), a leading candidate in gene therapy, has been proposed as a vaccine scaffold, but high production costs limit its use. Here we establish intracellular production of AAV VLPs in E...

pH-sensitive fluorescent proteins as genetically encoded pH sensors are promising tools for monitoring intra- and extracellular pH. However, there is a lack of ratiometric pH sensors, which offer a good dynamic range and can be purified and applied extracellularly to investigate uptake. In our study, the bright fluorescent protein CoGFP_V0 was C-te...

Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR designed ankyrin repeat protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to...

The development of peptide stapling techniques to stabilise α-helical secondary structure motifs of peptides led to the design of modulators of protein–protein interactions, which had been considered undruggable for a long time. We disclose a novel approach towards peptide stapling utilising macrocyclisation by late-stage Suzuki–Miyaura cross-coupl...

The vaccine ChAdOx1 nCoV-19 has been widely used, but its purity has been disputed because of rare side effects. We used Nanopore sequencing to assess the sequence and genetic purity of a vaccine dose. As we were lacking a reference sequence for the antigen cassette, we provide the obtained annotated sequence of the full vector to aid further studi...

Multiple, site-specific protein conjugation is increasingly attractive for the generation of antibody–drug conjugates (ADCs). As it is important to control the number and position of cargoes in an ADC, position-selective generation of reactive sites in the protein of interest is required. Formylglycine (FGly) residues are generated by enzymatic con...

Virus-like particles (VLPs) are increasingly used for vaccine development and drug delivery. Assembly of VLPs from purified monomers in a chemically defined reaction is advantageous compared to in vivo assembly, because it avoids encapsidation of host-derived components and enables loading with added cargoes. This review provides an overview of ex...

[This corrects the article DOI: 10.1093/nargab/lqaa074.].

An array of l- and d-halotryptophans with different substituents at the indole moiety was synthesized employing either enzymatic halogenation by halogenases or incorporation of haloindoles using tryptophan synthase. Introduction of these Trp derivatives into RGD peptides as a benchmark system was performed to investigate their influence on bioactiv...

The epidermal growth factor receptor (EGFR) plays a central role in the progression of many solid tumors. We used this validated target to analyze the de novo design of EGFR-binding peptides and their application for the delivery of complex payloads via rational design of a viral vector. Peptides were computationally designed to interact with the E...

Next-generation sequencing of single-stranded DNA (ssDNA) enables transgene characterization of gene therapy vectors such as adeno-associated virus (AAV), but current library generation uses complicated and potentially biased second-strand synthesis. We report that libraries for nanopore sequencing of ssDNA can be conveniently created without secon...

Formylglycine‐generating enzymes specifically oxidize cysteine within the consensus sequence CxPxR to C α ‐formylglycine (FGly). This non‐canonical electrophilic amino acid can subsequently be addressed selectively via bioorthogonal Hydrazino‐ iso ‐Pictet‐Spengler (HIPS) or Knoevenagel ligation to attach payloads like fluorophores or drugs to prote...

Monitoring DNA integrity and DNA contaminants in adeno-associated virus (AAV) gene therapy vectors is of major interest, because of clinical applications with increasing therapeutic doses. We here report direct, amplification-free nanopore sequencing of single-stranded AAV DNA using a rapid transposase-based protocol. Direct sequencing of bacteriop...

Research and clinical applications of recombinant adeno-associated virus (rAAV) significantly increased in recent years alongside regulatory approvals of rAAV gene therapy products. To date, all rAAV vectors as well as AAV empty capsids are produced in eukaryotic cells. We explored a new route to generate AAV capsids with the aim to analyze capsid...

Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stabilit...

Monomeric RGD peptides show an unspecific fluid‐phase uptake in cells, whereas multimeric RGD peptides are supposed to be internalized by integrin‐mediated endocytosis. However, a potential correlation between uptake mechanism and molecular mass has been neglected so far. A dual derivatization of peptide c (RGDw(7Br)K) was performed to investigate...

Formylglycine‐generating enzymes provide a convenient tool for site‐specific protein derivatization. Their ability to oxidize cysteine or serine residues within a defined consensus sequence to Cα‐formylglycine (FGly) allows for targeted introduction of a unique chemical handle for various bioconjugation reactions. In recent years, oxygen‐dependent...

Biosensors have emerged as a valuable tool with high specificity and sensitivity for fast and reliable detection of hazardous substances in drinking water. Numerous substances have been addressed using synthetic biology approaches. However, many proposed biosensors are based on living, genetically modified organisms and are therefore limited in she...

The epidermal growth factor receptor (EGFR) is a transmembrane protein involved in cell signaling processes and dysregulation of its activity often drives tumor growth. EGFR is a clinically validated tumor marker and target for antibodies and tyrosine kinase inhibitors. We demonstrate that a fusion protein of the natural ligand epidermal growth fac...

Formylglycine generating enzymes are of increasing interest in the field of bioconjugation chemistry. They catalyze the site‐specific oxidation of a cysteine residue to the aldehyde containing amino acid Cα‐formylglycine (FGly). This non‐canonical residue can be generated within any desired target protein and subsequently be used for bioorthogonal...

Formylglycine generating enzymes are of increasing interest in the field of bioconjugation chemistry. They catalyze the site‐specific oxidation of a cysteine residue to the aldehyde containing amino acid Cα‐formylglycine (FGly). This non‐canonical residue can be generated within any desired target protein and subsequently be used for bioorthogonal...

Strategies for site-specific modification of proteins have increased in number, complexity, and specificity over the last years. Such modifications hold the promise to broaden the use of existing biopharmaceuticals or to tailor novel proteins for therapeutic or diagnostic applications. The recent quest for next-generation antibody-drug conjugates (...

Protein design is usually limited to 20 amino acids. Since the chemical abilities of these amino acids are limited, a lot of interesting functions are not applicable in protein design. We aim to enable the translational incorporation of non-canonical amino acids (ncAAs) through an expanded genetic code. First, we repurposed the rarely used amber st...

For various experimental applications, microbial cultures at defined, constant densities are highly advantageous over simple batch cultures. Due to high costs, however, devices for continuous culture at freely defined densities still experience limited use. We have developed a small-scale turbidostat for research purposes, which is manufactured fro...

List of used commercial parts with suppliers. (DOCX)

3D printed parts as STL-files for printing. (ZIP)

Board schematic of custom board connected to the Arduino. The two MOSFETs (U1 and U2) handle the currents driving the pumps and the stirrer motor. Furthermore, the board connects to a pair of light-to-frequency converters, the laser and a Hall sensor mounted on the board, which measures the stirrer speed. (PDF)

Board layout for custom Arduino shield. (ZIP)

Firmware and graphical user interface files. (ZIP)

Linearity of optical density (OD) measurements performed in the turbidostat. Eight E. coli culture samples with optical densities between 0.024 and 1.982 were taken from the turbidostat and measured in a photometer at both 600 nm (blue squares) and 650 nm (red circles) in a 1 cm path length cuvette. Linear regressions for both wavelengths up to an...

Models of 3D printed parts. (A) Optics holder. (B) Tube holder. (C) Housing upper part. (D) Base plate. (E) Housing lower part. (F) Bottom cover. (G) Optics and tube holder assembled on base plate with board. (H) Fully assembled turbidostat central unit (4-pin connector to pumps not shown). (PNG)

Video clip of state estimator output for an E. coli BL21 culture, sped up by a factor of 10. (MP4)

We are exploring the application of unnatural base pairs (UBPs) as an expansion of the genetic code. To prevent loss of the unnatural base pair during reproduction we will utilize several systems including CRISPR/Cas9. The extended genetic code allows the ribosomal incorporation of several non-canonical amino acids (ncAAs) into peptides. With their...

Evobodies - molecular speed dating We developed a novel and easy to use system for the generation of binding proteins in E. coli via in vivo directed evolution. Resulting proteins called Evobodies have the potential to bind specific to target proteins enabling various medical and analytical applications. Big advantages of our low-cost system are th...

Introduction: Epidermal growth factor receptor (EGFR) expression is upregulated in many tumors and its aberrant signaling drives progression of many cancer types. Consequently, EGFR has become a clinically validated target as extracellular tumor marker for antibodies as well as for tyrosine kinase inhibitors. Within the last years, new mechanistic...

D-peptides have been attributed pharmacological advantages over regular L-peptides, yet design rules are largely unknown. Based on a designed coiled-coil like D/L heterotetramer, named L-Base/D-Acid, we generated a library offering alternative residues for interaction with the D-peptide. Phage display selection yielded one predominant peptide, name...

The non-random chromosomal translocations t(10;11)(p13;q23) and t(10;11)(p13;q14-21) result in leukemogenic fusion proteins comprising the coiled coil domain of the transcription factor AF10 and the proteins MLL or CALM, respectively, and subsequently cause certain types of acute leukemia. The AF10 coiled-coil domain, which is crucial for the leuke...

Understanding and controlling proteolysis is an important goal in therapeutic chemistry. Among the natural products specifically inhibiting proteases microviridins are particularly noteworthy. Microviridins are ribosomally produced and posttranslationally modified peptides that are processed into a unique, cagelike architecture. Here, we report a c...

Ein wichtiges Ziel in der therapeutischen Chemie ist das Verständnis und die Kontrolle von proteolytischen Prozessen. Unter den proteaseinhibierenden Naturstoffen nehmen Microviridine eine Sonderstellung ein. Microviridine sind ribosomal erzeugte und posttranslational modifizierte Peptide, die zu einer einzigartigen Struktur mit käfigartiger Gestal...

The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these...

DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning. A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucle...

SIRT6 is a NAD+-dependent deacetylase that modulates chromatin structure and safeguards genomic stability. So far, SIRT6 has been assigned to the nucleus and only nuclear targets of SIRT6 are known. Here, we demonstrate that in response to stress, C. elegans SIR-2.4 and its mammalian orthologue SIRT6 localize to cytoplasmic stress granules (SGs), i...

The twin-arginine translocation (TAT) pathway of the bacterial cytoplasmic membrane mediates translocation only of proteins that accomplished a native-like conformation. We deploy this feature in modular selection systems for directed evolution, in which folding helpers as well as dimeric or oligomeric protein-protein interactions enable TAT-depend...

Management of data to produce scientific knowledge is a key challenge for biological research in the 21st century. Emerging high-throughput technologies allow life science researchers to produce big data at speeds and in amounts that were unthinkable just a few years ago. This places high demands on all aspects of the workflow: from data capture (i...

The stability of proteins is paramount for their therapeutic and industrial use and, thus, is a major task for protein engineering. Several types of chemical and physical stabilities are desired, and discussion revolves around whether each stability trait needs to be addressed separately and how specific and compatible stabilizing mutations act. We...

Synthetic Biology is founded on the idea that complex biological systems are built most effectively when the task is divided in abstracted layers and all required components are readily available and well-described. This requires interdisciplinary collaboration at several levels and a common understanding of the functioning of each component. Stand...

Remote control of cells is a desirable feature in synthetic biology. We established a light-switchable interfering peptide (iPEP) which controls gene expression by modulating the activity of a transcription factor. For photo-switching, the iPEP is cross-linked with a cis-trans isomerizable cross-linker in such a way that the light-activated cis for...

Phage display with filamentous phages is widely applied and well developed, yet proteins requiring a cytoplasmic environment for correct folding still defy attempts at functional display. To extend applicability of phage display, we employed the twin-arginine translocation (TAT) pathway to incorporate proteins fused to the C-terminal domain of the...

Remarkable success in optimizing complex properties within DNA and proteins has been achieved by directed evolution. In contrast to various random mutagenesis methods and high-throughput selection methods, the number of available DNA shuffling procedures is limited, and protocols are often difficult to adjust. The strength of the nucleotide exchang...

Synthetic Biology is advanced by many users and relies on the assembly of genetic elements to devices, systems and finally genomes. SynBioWave is a software suite that enables multiple distributed users to analyze and construct genetic parts in real-time collaboration. It builds on Google Wave and provides an extensible robot-robot-user communicati...

Systematic tandem-affinity-purification (TAP) of protein complexes was tremendously successful in yeast and has changed the general concept of how we understand protein function in eukaryotic cells. The transfer of this method to other model organisms has been difficult and may require specific adaptations. We were especially interested to establis...

Synthetic biology is an emerging field with large potential for research and development, and future benefits for economy and society. The European Union (EU) has started measures to structure and develop the field, such as a high‐level expert group. However, research activities are still scattered across Europe and scientific disciplines, and are...

This Request for Comments (RFC) describes an extension to the original BioBrick assembly standard (BBF RFC 10). The Fusion Assembly strategy described here is fully backwards compatible with RFC 10 and supports the construction of in frame fusion proteins.

We have employed semirational design in combination with selection systems to generate peptides interfering with a pathogenic protein-protein interaction: the transcriptional regulator Activator Protein-1 (AP-1). Peptide inhibitors with high interaction stability were screened and selected using either phage display or protein-fragment complementat...

Cell physiology depends on a fine-tuned network of protein-protein interactions, and misguided interactions are often associated with various diseases. Consequently, peptides, which are able to specifically interfere with such adventitious interactions, are of high interest for analytical as well as medical purposes. One of the most abundant protei...

Semi-rational design is combined with PCAs (protein-fragment complementation assays) and phage-display screening techniques to generate a range of iPEPs (interfering peptides) that target therapeutically relevant proteins with much higher interaction stability than their native complexes. PCA selection has been improved to impose a competitive and...

The activator protein-1 (AP-1) complex plays a crucial role in numerous pathways, and its ability to induce tumorigenesis is well documented. Thus, AP-1 represents an interesting therapeutic target. We selected peptides from phage display and compared their ability to disrupt the cFos/cJun interaction to a previously described in vivo protein-fragm...

c-Myc is one of the most frequently deregulated oncogenes in human cancers, and recent studies showed that even brief inactivation of Myc can be sufficient to induce tumor regression or loss. Consequently, inactivation of Myc provides a novel therapeutic opportunity and challenge, as the dimerization of Myc with Max is crucial for its function. We...

Relating primary sequence to three-dimensional structure has long been the holy grail of structural biology and appears to be far from achievement. Within grasp however, is the use of intuitive or unintuitive methodology to modify existing known protein structures to achieve the desired effect. We use protein engineering as a general term for the d...

Enzymes can dramatically increase the rate of chemical reactions while acting stereoselective and regioselective and are therefore very attractive for industry. However, only few enzymes taken from nature work under the often harsh conditions required by industrial applications. Proteins with desired attributes can be obtained either by searching t...

The energetic determinants that drive specific protein-protein interactions are not entirely understood. We describe simultaneous in vivo selection of specific and stable interactions using homologous peptides which compete with protein libraries for an interaction with a target molecule. Library members binding to their target, and promoting cell...

Coiled coil motifs are, despite their apparent simplicity, highly specific, and play a significant role in the understanding of tertiary structure and its formation. The most commonly observed of the coiled coils, the parallel dimeric, is yet to be fully characterized for this structural class in general. Nonetheless, strict rules have emerged for...

Mimicking natural evolution by DNA shuffling is a commonly used method for the optimization of DNA and protein properties. Here, we present an advancement of this approach whereby a gene library is amplified using a standard polymerase chain reaction (PCR), but incorporates dUTP as a fragmentation-defining exchange nucleotide, together with the fou...

Improving enzyme stability is a highly desirable design step in generating enzymes able to function under extreme conditions, such as elevated temperatures, while having the additional benefit of being less susceptible to cleavage by proteases. For these reasons, many different approaches and techniques have been devised in constructing such protei...

Activator protein-1 (AP-1) is a crucial transcription factor implicated in numerous cancers. For this reason, nine homologues of the AP-1 leucine zipper region have been characterized: Fos (c-Fos, FosB, Fra1, and Fra2), Jun (c-Jun, JunB, and JunD), and semirational library-designed winning peptides FosW and JunW. The latter two were designed to spe...