Andre Koltermann

A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins

GFP and the red fluorescent protein, DsRed, have been combined to design a protease assay that allows not only for fluorescence resonance energy transfer (FRET) studies but also for dual-color crosscorrelation analysis, a single-molecule-based method that selectively probes the concomitant movement of two distinct tags. The measurement principle is based on a spectrally resolved detection of single molecules diffusing in and out of a diffraction-limited laser focus. Double-labeled substrate molecules are separated into two single-labeled products by specific cleavage at a protease cleavage site between the two flanking tags, DsRed and GFP, thus disrupting joint fluctuations in the two detection channels and terminating FRET between the two labels. In contrast to enzyme assays based solely on FRET, this method of dual-color crosscorrelation is not limited to a certain range of distances between the fluorophores and is much more versatile with respect to possible substrate design. To simplify the measurement setup, two-photon excitation was used, allowing for simultaneous excitation of both tags with a single infrared laser wavelength. The general concept was experimentally verified with a GFP–peptide–DsRed construct containing the cleavage site for tobacco etch virus protease. Two-photon excitation in the infrared and the use of cloneable tags make this assay easily adaptable to intracellular applications. Moreover, the combination of FRET and crosscorrelation analysis in a single-molecule-based approach promises exciting perspectives for miniaturized high-throughput screening based on fluorescence spectroscopy.

Cell‐Free Metabolic Engineering: Production of Chemicals by Minimized Reaction Cascades

The limited supply of fossil resources demands the development of renewable alternatives to petroleum-based products. Here, biobased higher alcohols such as isobutanol are versatile platform molecules for the synthesis of chemical commodities and fuels. Currently, their fermentation-based production is limited by the low tolerance of microbial production systems to the end products and also by the low substrate flux into cell metabolism. We developed an innovative cell-free approach, utilizing an artificial minimized glycolytic reaction cascade that only requires one single coenzyme. Using this toolbox the cell-free production of ethanol and isobutanol from glucose was achieved. We also confirmed that these streamlined cascades functioned under conditions at which microbial production would have ceased. Our system can be extended to an array of industrially-relevant molecules. Application of solvent-tolerant biocatalysts potentially allows for high product yields, which significantly simplifies downstream product recovery.

PRINCIPLES AND METHODS OF EVOLUTIONARY BIOTECHNOLOGY

Evolutionary biotechnology applies the principles of molecular evolution to biotechnology, leading to novel techniques for the creation of biomolecules with a great variety of functions for technical and medical purposes. Several basic principles for the application of evolutionary strategies can be derived from a comprehensive theory of molecular evolution. Prerequisites for evolutionary biotechnology are summarized with respect to the different classes of biomolecules and a few, selected applications are described in detail. Concepts for the technical implementation of evolutionary strategies are presented which allow automatized, high throughput processes.

Evolutionary Biotechnology - Reflections and Perspectives

With the designing of biomolecules, a new era in biotechnology has been initiated. One of the most promising strategies for successfully designing complex biomolecular functions is to exploit nature’s principles of Darwinian evolution, i.e. variation and selection. The application of these principles to directed evolution of molecules is the underlying concept of evolutionary biotechnology. Important prerequisites are a comprehensive understanding of the mode of molecular evolution as well as the ability to apply its principles to experimental systems in order to create and optimize molecular functions with scientific or economic value.

High throughput screening by multichannel glass fiber fluorimetry

As a tool for screening large numbers of biological samples by means of amplification (e.g., Qβ or PCR) we have constructed a thermocycler that includes optionally a 96-channel or 960-channel glass fiber fluorimeter (combined with a cooled charge-coupled-device camera). We briefly describe the system integration of all components like liquid handling, thermostats, an x,y,z robot arm, and the glass fiber fluorimeter. The integrated glass fiber fluorimeter allows sensitive on-line measurements in 960 channels within 5 s. Two different screening procedures were carried out. In a first experiment PCR reactions were done in the presence of the known PCR inhibitor hematin and its suppressor transferrin. The system was used to titrate the suppressor with the inhibitor hematin in order to determine the maximum inhibitor concentration tolerated at a given suppressor concentration. We processed 96 PCR samples in parallel with 11 different concentration steps. In a second experiment the 960-channel glass fiber fluor...

Production of human interleukin-8 expressed in Escherichia coli: From a laboratory scale for in vitro tests via a technical scale for animal studies to a process scale for a GMP-compatible production

An Escherichia coli K 12 strain has been constructed for efficient expression of recombinant biologically active human IL-8 (Interleukin-8). The development of a fermentation and purification process from the laboratory scale (cells from 15 l fermentation broth) to a production scale (cells from 200 l fermentation broth) is described. Material obtained from the laboratory scale was used for initial in vitro studies and for the development of a biological assay. An upscale purification process starting from 80 l fermentation broth resulted in larger amounts of IL-8 needed for preclinical studies. This process includes a fully automated control of the initial affinity chromatography step. Finally, a production process which differed markedly from the small-scale processes was tailor-made for GMP conformity and economic considerations. It consists of a cell disruption step followed by two crossflow diafiltrations with different molecular weight cut offs and filtration rates, one cation exchange chromatography and a final dialysis step. In order to enhance the overall yield of biologically active IL-8, conditions for a resolubilisation of insoluble IL-8 present in the remaining pellet after cell disruption were worked out.

Dual-Color Confocal Fluorescence Spectroscopy and its Application in Biotechnology

In recent years, fluorescence correlation spectroscopy (FCS) has become an attractive analytical tool for the investigation of biomolecular processes at the single molecular level. This method was invented in the early 1970s by groups at Cornell University, Ithaca, N.Y. [9.1,9.2], and at the Karolinska Institute, Stockholm [9.3,9.4]. During the 1990s, modern confocal optics, new dyes as efficient fluorescent probes, sensitive photon detectors, and fast data processing tools have been introduced, mainly by Rigler and Eigen [9.5,9.6]. Due to these improvements, FCS permits the observation of the dynamics of single molecules in real time while they pass an open volume element of less than a femtoliter, i.e. the size of a common bacterial cell. Nowadays, FCS has found its way into several laboratories and companies all over the world as a tool for basic research as well as for industrial applications such as drug screening.