Kristian M. Müller

High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions

Abstract Functional bivalent miniantibodies, directed against the epidermal growth factor receptor, accumulated to more than 3 gl−1 in high-cell-density cultures of Escherichia coli RV308(pHKK) on a pilot scale. The miniantibodies consist of scFv fragments with a C-termi-nal hinge followed by a helix-turn-helix motif, which homodimerizes in vivo. The improved expression vector pHKK is characterized by the hok/sok suicide system, improving plasmid maintenance, and the inducible lac p/o promoter system with the very strong T7g10 Shine-Dalgarno sequence. The expression unit is flanked by terminators. The prototrophic RV308 cells were cultivated in glucose mineral salt medium and reached a cell density of 145 g dry biomass l−1 after 33 h. After induction, growth continued almost unchanged for a further 4 h with concomitant miniantibody formation. In the fed-batch phase, the concentration of glucose was kept almost constant at the physiological level of approximately 1.5 g l−1, using on-line flow injection analysis for control. Surprisingly, E. coli RV308(pHKK) did not accumulate significant amounts of the metabolic by-product acetate under these unlimited aerobic growth conditions.

THE FIRST CONSTANT DOMAIN (CH1 AND CL) OF AN ANTIBODY USED AS HETERODIMERIZATION DOMAIN FOR BISPECIFIC MINIANTIBODIES

Bispecific miniantibodies were constructed by genetically fusing the CH1 domain of an IgG1 to the C‐terminus of a single‐chain Fv fragment (scFv‐425), specific for the EGF receptor, and fusing the CL domain of a kappa light chain to the C‐terminus of a scFv specific for CD2 (scFv‐M1). An efficient dicistronic gene arrangement for functional expression in Escherichia coli was constructed. Immunoblots demonstrated correct domain assembly and the formation of the natural CH1‐CL disulfide bridge. Gel filtration confirmed the correct size, sandwich ELISAs demonstrated bispecific functionality, and SPR biosensor measurements determined binding to EGF‐R in comparison to bivalent constructs. Bispecific anti‐EGF‐R/anti‐CD2 miniantibodies are candidates for the immunotherapy of cancer.

A dimeric bispecific miniantibody combines two specificities with avidity

Bispecific antibodies extend the capabilities of nature and might be applied in immunotherapy and biotechnology. By fusing the gene of a single‐chain Fv (scFv) fragment to a helical dimerization domain, followed by a second scFv fragment of different specificity, we were able to express a functional protein in E. coli, which is bispecific and has two valencies for each specificity. The dimeric bispecific (DiBi) miniantibody preserves the natural avidity of antibodies in a very small‐sized molecule of only 120 kDa. The generality of the principle was shown with a scFv fragment binding the EGF‐receptor (named scFv 425) in three combinations with scFv fragments either directed against CD2 (ACID2.M1), phosphorylcholine (McPC603) or fluorescein (FITC‐E2). Binding was analyzed by sandwich surface plasmon resonance biosensor (BIAcore) measurements.

Selectively infective phage (SIP) technology: scope and limitations

We review here the selectively infective phage (SIP) technology, a powerful tool for the rapid selection of protein-ligand and peptide-ligand pairs with very high affinities. SIP is highly suitable for discriminating between molecules with subtle stability and folding differences. We discuss the preferred types of applications for this technology and some pitfalls inherent in the in vivo SIP method that have become apparent in its application with highly randomized libraries, as well as some precautions that should be taken in successfully applying this technology.

Standardization in synthetic biology.

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. Standardization of the physical composition and the description of each part is required as well as a controlled vocabulary to aid design and ensure interoperability. Here, we describe standardization initiatives from several disciplines, which can contribute to Synthetic Biology. We provide examples of the concerted standardization efforts of the BioBricks Foundation comprising the request for comments (RFC) and the Registry of Standardized Biological parts as well as the international Genetically Engineered Machine (iGEM) competition.

Workshop on laboratory protocol standards for the molecular methods database

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 (including the experimental constraints of the experiment), analysis and preservation, to peer-reviewed publication of results. Failure to recognise the issues at each level can lead to serious conflicts and mistakes; research may then be compromised as a result of the publication of non-coherent protocols, or the misinterpretation of published data. In this report, we present the results from a workshop that was organised to create an ontological data-modelling framework for Laboratory Protocol Standards for the Molecular Methods Database (MolMeth). The workshop provided a set of short- and long-term goals for the MolMeth database, the most important being the decision to use the established EXACT description of biomedical ontologies as a starting point.

Quality assurance for biopharmaceuticals: An overview of regulations, methods and problems

Abstract This paper provides an overview of regulations with special regard to Good Manufacturing Practice (GMP) of biopharmaceuticals in different countries (Switzerland, European Union, USA). Problems during the set-up and maintenance of a cell bank and expression system, fermentation, protein purification, and product testing are outlined as well as methods and techniques for quality assurance during these processes. Freeze-drying and aseptic filling of the finished dosage form are discussed.

Light-controlled gene switches in mammalian cells.

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 form enables inhibitor folding rendering it active, whereas the dark-adapted trans form forces the inhibitor into an inactive form. Switching can be repeated in both directions. The iPEP acts as dominant-negative inhibitor targeting c-Jun and c-Fos of the transcription factor activator protein-1 (AP-1). Light-activated peptides exhibited much stronger inhibition of AP-1:DNA complexes and interference with gene transcription than their nonactivated counter parts. In this chapter, we provide protocols for cross-linking, peptide purification, observation of structural changes upon photo-switching, DNA binding analyses as well as gene expression studies in mammalian cells.