Purification Of Target Protein Research Articles

Parallel reaction monitoring targeted mass spectrometry as a fast and sensitive alternative to antibody-based protein detection

The reliable, accurate and quantitative targeted detection of proteins is a key technology in molecular and cell biology and molecular diagnostics. The current golden standard for targeted protein detection in complex mixtures such as complete cell lysates or body fluids is immunoblotting, a technology that was developed in the late 1970s and has not undergone major changes since. Although widespread, this methodology suffers from several disadvantages, such as the inability to detect low-abundant proteins or specific posttranslational modifications, the requirement for highly specific antibodies, the lack of quantitative power and the often-tedious practical procedures. Mass spectrometry (MS) based targeted protein detection is an alternative technology that could circumvent these caveats. Here, we compare immunoblotting with targeted protein mass spectrometry using a parallel reaction monitoring (PRM) regime on the Orbitrap mass spectrometer. We show that PRM based MS has superior sensitivity and quantitative accuracy over immunoblotting. The limit of detection for proteolytic peptides of a purified target protein was found to be in the mid- to low-attomole range and approximately one order of magnitude higher when embedded in a complex biological matrix. The incorporation of synthetic heavy isotope labeled (AQUA) peptides as internal calibrants into the PRM workflow allows for even higher accuracy for both the relative and absolute quantitation of tryptic target peptides. In conclusion, PRM is a versatile and sensitive technology, which can overcome the shortcomings of immunoblotting. We argue that PRM based MS could become the method of choice for the targeted detection of proteins in complex cellular matrices or body fluids and may eventually replace standard methods such as Western blotting and ELISA in biomedical research and in the clinic.

An immobilized antibody-based affinity grid strategy for on-grid purification of target proteins enables high-resolution cryo-EM

In cryo-electron microscopy (cryo-EM), sample preparation poses a critical bottleneck, particularly for rare or fragile macromolecular assemblies and those suffering from denaturation and particle orientation distribution issues related to air-water interface. In this study, we develop and characterize an immobilized antibody-based affinity grid (IAAG) strategy based on the high-affinity PA tag/NZ-1 antibody epitope tag system. We employ Pyr-NHS as a linker to immobilize NZ-1 Fab on the graphene oxide or carbon-covered grid surface. Our results demonstrate that the IAAG grid effectively enriches PA-tagged target proteins and overcomes preferred orientation issues. Furthermore, we demonstrate the utility of our IAAG strategy for on-grid purification of low-abundance target complexes from cell lysates, enabling atomic resolution cryo-EM. This approach greatly streamlines the purification process, reduces the need for large quantities of biological samples, and addresses common challenges encountered in cryo-EM sample preparation. Collectively, our IAAG strategy provides an efficient and robust means for combined sample purification and vitrification, feasible for high-resolution cryo-EM. This approach holds potential for broader applicability in both cryo-EM and cryo-electron tomography (cryo-ET).

Synthesis of biologically active proteins as L6KD-SUMO fusions forming inclusion bodies in Escherichia coli.

A new platform has been developed to facilitate the production of biologically active proteins and peptides in Escherichia coli. The platform includes an N-terminal self-associating L6 KD peptide fused to the SUMO protein (small ubiquitin-like protein modifier) from the yeast Saccharomyces cerevisiae, which is known for its chaperone activity. The target proteins are fused at the C termini of the L6 KD-SUMO fusions, and the resulting three-component fusion proteins are synthesized and self-assembled in E. coli into so-called active inclusion bodies (AIBs). In vivo, the L6 KD-SUMO platform facilitates the correct folding of the target proteins and directs them into AIBs, greatly simplifying their purification. In vitro, the platform facilitates the effective separation of AIBs by centrifugation and subsequent target protein release using SUMO-specific protease. The properties of the AIBs were determined using five proteins with different sizes, folding efficiencies, quaternary structure, and disulfide modifications. Electron microscopy shows that AIBs are synthesized in the form of complex fibrillar structures resembling "loofah sponges" with unusually thick filaments. The obtained results indicate that the new platform has promising features and could be developed to facilitate the synthesis and purification of target proteins and protein complexes without the use of renaturation.

Geldanaycin-encapsulated magnetic nanoparticle for isolation of myosin in proteomics

The grafting of a drug molecule, i.e., geldanamycin (GA) onto polyethyleneimine (PEI)-coated magnetic nanoparticle produces a novel composite, GA@Fe3O4–NH2. The composite is confirmed by characterizations with FT-IR, Raman, SEM, EDS, VSM and TEM. Due to the high binding-affinity of GA with myosin heavy chain (MYH), GA@Fe3O4–NH2 exhibits excellent adsorption performance towards myosin. Consequently, a solid-phase extraction procedure is established for highly efficient and selective separation of myosin from pig heart extract. At pH 6.0, an adsorption efficiency of 97.1 % is achieved for treating 100 μg mL−1 myosin (0.1 mL) with 0.1 mg GA@Fe3O4–NH2 as adsorbent. The adsorption behavior of myosin onto GA@Fe3O4–NH2 fits Langmuir model, corresponding to a theoretical adsorption capacity of 518.1 mg g−1. The adsorbed myosin can be readily recycled by the SDS solution (1 %, m/m) with an elution efficiency of 91.8 %. According to circular dichroism spectroscopy, the conformational changes of myosin during adsorption and elution are reversible. For practical application, myosin is successfully isolated from the pig left ventricular protein extract with GA@Fe3O4–NH2, and SDS-PAGE and LC-MS/MS showed that myosin had high purity and a total of 716 proteins could be identified. Significantly, Geldamycin-encapsulated magnetic nanoparticle for the separation of myosin well exploits the potential of the nanomaterials modified by drug molecules in the separation and purification of target proteins.

Synthesis of micron-sized magnetic agarose beads chelated with nickel ions towards the affinity-based separation of histidine-tagged/rich proteins

Developing high-performance magnetic particles for the effective separation and purification of target proteins has become an important topic in the area of biomedical research. In this work, a simple and novel strategy was proposed for fabricating magnetic Fe3O4@agarose-iminodiacetic acid-Ni microspheres (MAIN), which can efficiently and selectively isolate histidine-tagged/rich proteins (His-proteins). Based on the thermoreversible sol-gel transition of agarose, basic magnetic agarose microspheres were prepared through the inverse emulsion method, in which the emulsion contained agarose and amine-modified Fe3O4 nanoparticles. The size of the emulsion was controlled by the emulsification of a high-speed shear machine, which improved the specific surface area of MAIN. Subsequently, the amine-modified Fe3O4 nanoparticles were covalently crosslinked with agarose through epichlorohydrin, which could avoid leakage of the magnetic source during use and increase the stability of MAIN. The microsized MAIN exhibited a clearly visible spherical core-shell structure with a diameter range from 3.4 μm to 9.8 μm, and excellent suspension ability in aqueous solution. The maximum adsorption capacity of MAIN for histidine-rich bovine hemoglobin was 1069.2 mg g−1 at 35 °C, which was higher than those of commercialized and most reported magnetic agarose microspheres/nanoparticles. The MAIN showed excellent adsorption ability and selectivity toward His-proteins in a mixture of histidine-rich bovine serum albumin (BSA) and histidine-poor lysozyme (LYZ). When the amount of LYZ was 5-fold higher than that of BSA, the recovery of BSA reached 75.0%. To prove its practicability, MAIN was successfully employed for the enrichment of histidine-tagged RSV-F0 from the cell culture medium supernatant. According to the optimized conditions, MAIN could enrich approximately 0.1 mg of RSV-F0 from 1 mL of complex biological sample. Therefore, we believe that the novel MAIN could be applicable for efficient separation and purification of His-proteins from complex biological systems.

“Lab of the Future”─Today: Fully Automated System for High-Throughput Mass Spectrometry Analysis of Biotherapeutics

Here we describe a state-of-the-art, integrated, multi-instrument automated system designed to execute methods involved in mass spectrometry characterization of biotherapeutics. The system includes liquid and microplate handling robotics and utilities, integrated LC-MS, along with data analysis software, to perform sample purification, preparation, and analysis as a seamless integrated unit. The automated process begins with tip-based purification of target proteins from expression cell-line supernatants, which is initiated once the samples are loaded onto the automated system and the metadata are retrieved from our corporate data aggregation system. Subsequently, the purified protein samples are prepared for MS, including deglycosylation and reduction steps for intact and reduced mass analysis, and proteolytic digestions, desalting, and buffer exchange via centrifugation for peptide map analysis. The prepared samples are then loaded into the LC-MS instrumentation for data acquisition. The acquired raw data are initially stored on a local area network storage system that is monitored by watcher scripts that then upload the raw MS data to a network of cloud-based servers. The raw MS data are processed with the appropriately configured analysis workflows such as database search for peptide mapping or charge deconvolution for undigested proteins. The results are verified and formatted for expert curation directly in the cloud. Finally, the curated results are appended to sample metadata in the corporate data aggregation system to accompany the biotherapeutic cell lines in subsequent processes.

A histidine-rich fusion tag enables real-time monitoring of recombinant protein expression by Pauly reaction-based colorimetric assay

Commercially available recombinant expression systems always use fusion tags to facilitate target protein purification and SDS-PAGE analysis followed by Coomassie Brilliant Blue (CBB) staining is the classical method to validate the expression level of target protein, which is time-consuming, although not very laborious. Previously, we found that a histidine-rich elastin-like polypeptide (HRELP) tag could make its fusion proteins being quickly and specifically stained with Pauly's reagent. In this study, we designed a Pauly reaction-based colorimetric assay to real-time monitoring of the expression level of recombinant protein tagged HRELP and found that the absorption value of post-induction E. coli cells stained with Pauly's reagent correlated well with both the band intensity of the target protein from Pauly's reagent-stained and CBB-stained gels. Moreover, we found the colorimetric assay could also be helpful to roughly estimate the expression efficiency by using a poly-histidine-tagged protein, which has only 1.17% histidine residue. In our opinion, Pauly reaction-based colorimetric assay could significantly shorten the time to validate the over-expression of recombinant protein tagged with either HRELP or poly-histidine. And HRELP seemed to be an ideal fusion tag for it can not only facilitate protein purification but also simplify protein detection.

In vitro production of N-degron fused proteins and its application.

The N-degron pathway, first discovered several decades ago by Varshavsky's laboratory, controls the half-life of target proteins depending on their N-terminal residues. In vivo cell biology studies have established the physiological role of the N-degron pathway. However, in vitro studies such as biochemical assays and structural biology studies are relatively limited. The N-degron substrates cannot be obtained via simple protein expression. The N-degron residues are exposed via the proteolytic process from the translated nascent polypeptide chains. Thus, methods for the fusion expression with several cleavable tags and subsequent treatment with specific proteases to design the exposed N-degron signals have been introduced. Recently, we developed a unique fusion technique using microtubule-associated protein 1A/1B light chain 3B (LC3B), a key marker protein of autophagy, to obtain a high yield of the purified target proteins with variable N-terminal residues for various biochemical studies including enzymatic and binding assays, and crystallization of N-degron complex. This chapter describes the protocols that include the vector map designed for producing LC3B fused target proteins, methods for expression and purification of an example protein, p62/SQSMT1, using different N-terminal residues, and methods to obtain the purified ATG4B protease, which is used for processing LC3B tag and exposing the required N-terminal residues of the target protein.

Optimising expression and extraction of recombinant proteins in plants.

Recombinant proteins are of paramount importance for research, industrial and medical use. Numerous expression chassis are available for recombinant protein production, and while bacterial and mammalian cell cultures are the most widely used, recent developments have positioned transgenic plant chassis as viable and often preferential options. Plant chassis are easily maintained at low cost, are hugely scalable, and capable of producing large quantities of protein bearing complex post-translational modification. Several protein targets, including antibodies and vaccines against human disease, have been successfully produced in plants, highlighting the significant potential of plant chassis. The aim of this review is to act as a guide to producing recombinant protein in plants, discussing recent progress in the field and summarising the factors that must be considered when utilising plants as recombinant protein expression systems, with a focus on optimising recombinant protein expression at the genetic level, and the subsequent extraction and purification of target proteins, which can lead to substantial improvements in protein stability, yield and purity.

Facile synthesis of novel helical imprinted fibers based on zucchini-derived microcoils for efficient recognition of target protein in biological sample

Highly selective recognition and purification of target proteins from complex biological matrices remains a challenging subject in natural and life sciences. Compared with natural recognition receptors, artificial imprinted polymers are an ideal alternative candidate. In this study, we report a novel method to prepare helical protein imprinted fibers (HPIFs) with zucchini-derived microcoils as a carrier, firstly. Inspired by the self-polymerization of adhesive proteins in mussels, dopamine and 3,4-dihydroxyphenylacetic acid were chosen as bifunctional monomers for the first time to form a biocompatible imprinted layer. The chemical/physical properties and recognition performance of HPIFs were studied in a series of experiments. Additionally, the practicability of HPIFs was verified by specifically recognizing target protein in complex egg white sample. The one-step synthesis process and excellent binding performance of HPIFs make them a promising material for protein recognition and purification, and endow HPIFs with potential application value in the food, chemical and pharmaceutical fields.

Fabrication of copolymer brushes grafted superporous agarose gels: Towards the ultimate ideal particles for efficient affinity chromatography

A composite immobilized-metal affinity agarose particle was designed for the selective separation and purification of histidine-tagged proteins from complicated biological samples. The composite particle was constructed using superporous agarose particles as supporting matrix, flexible copolymer brushes as scaffolds to render higher ligand densities, and Ni2+-chelated iminodiacetic acids as recognition elements. Superporous agarose composite particles endow high permeability and interfering substance tolerance. The copolymer brush was prepared by surface-initiated atom transfer radical polymerization of N-isopropylacrylamide and glycidyl methacrylate, followed by iminodiacetic acids and Ni2+ ions. The physical and chemical properities of the composite particle were thoroughly investigated. The composite particles were shown to be able to selectively separate histidine-tagged recombinant proteins in the presence of high quantities of interfering chemicals in a model protein-binding experiment. By altering the temperature, the protein binding of the composite particles can be modulated. The superporous agarose particles supported polymer brush enables fast and efficient separation and purification of target proteins with high permeability, low backpressure, and high interfering matrix tolerance, which pave the path for bioseparation through designing and fabrication of novel agarose particles-based functional materials.

Affinity of phenolic compounds for transition metal ions immobilized on cation-exchange columns

Immobilized metal ion affinity chromatography (IMAC) is useful in purification of histidine-tagged or histidine-rich proteins and peptides from a variety of hosts. However, phenolic compounds including polyphenols interfere with IMAC due to their high affinities for the transition metals immobilized on the column resins, which hampers the purification of proteins from plant-based host systems. In contrast to extensive knowledge of the mechanism of the interactions between phenolic compounds and transition metal ions in solution, an understanding of the interactions on the columns, where transition metal ions are immobilized on the resins, remains elusive. This study systematically investigated the affinity of phenolic compounds for transition metal ions by varying the number and position of phenolic hydroxyl groups (OH groups) and using different transition metals—Fe(II), Cu(II) and Ni(II)—on various IMACs, in which the columns were fabricated by equilibrating the cation-exchange column with transition metal solutions. It was found that the more OH groups the aromatic compounds have, the higher the affinity for transition metal ions; in particular, methyl gallate and pyrogallol were permanently bound to the IMAC column, which reflected coordinate bond formation with the transition metal ions. Importantly, the phenolic compounds showed no obvious affinity for the Ni(II)-IMAC column, in contrast to the Fe(II)- and Cu(II)-IMAC columns, whereas imidazole and histidine-tagged proteins showed evident binding to the Ni(II)-IMAC column. Ni(II)-IMAC should thus be especially effective in isolating histidine-tagged and histidine-rich species from phenolic compound-containing systems. These results indicate that the affinity between phenolic compounds and transition metal ions on the column is consistent with the results in solution. They also provide a comprehensive view for devising strategies to improve IMAC purification of target proteins and peptides from samples containing phenolic compounds.

Accelerated strain construction and characterization of C. glutamicum protein secretion by laboratory automation

Secretion of bacterial proteins into the culture medium simplifies downstream processing by avoiding cell disruption for target protein purification. However, a suitable signal peptide for efficient secretion needs to be identified, and currently, there are no tools available to predict optimal combinations of signal peptides and target proteins. The selection of such a combination is influenced by several factors, including protein biosynthesis efficiency and cultivation conditions, which both can have a significant impact on secretion performance. As a result, a large number of combinations must be tested. Therefore, we have developed automated workflows allowing for targeted strain construction and secretion screening using two platforms. Key advantages of this experimental setup include lowered hands-on time and increased throughput. In this study, the automated workflows were established for the heterologous production of Fusarium solani f. sp. pisi cutinase in Corynebacterium glutamicum. The target protein was monitored in culture supernatants via enzymatic activity and split GFP assay. Varying spacer lengths between the Shine-Dalgarno sequence and the start codon of Bacillus subtilis signal peptides were tested. Consistent with previous work on the secretory cutinase production in B. subtilis, a ribosome binding site with extended spacer length to up to 12 nt, which likely slows down translation initiation, does not necessarily lead to poorer cutinase secretion by C. glutamicum. The best performing signal peptides for cutinase secretion with a standard spacer length were identified in a signal peptide screening. Additional insights into the secretion process were gained by monitoring secretion stress using the C. glutamicum K9 biosensor strain.Key points• Automated workflows for strain construction and screening of protein secretion• Comparison of spacer, signal peptide, and host combinations for cutinase secretion• Signal peptide screening for secretion by C. glutamicum using the split GFP assay

Modification of C‐segment of CfaDnaE split intein for improving clean‐in‐place in chromatography process

This research focuses on the construction of an affinity purification system based on Cfa DnaE split intein. Cfa DnaE intein is an artificially constructed intein with the advantages of a fast cleavage reaction and good stability. In a previous study, a purification system that uses Cfa intein as a tag was constructed, the separation of the target protein and the tag during the purification process was completed, and the purity of the purified target protein reached 98.21%. Guided by molecular docking results, we identified flexible regions in the split intein and inserted several glycines into the protein to decrease the stability of the Cfa IC , thereby improving the regenerability of the IN media. Inserting 6 glycines between amino acids 14 and 15 of IC improved the regeneration rate of IC -GFP on the column to approximately 96%.

C9Mab-1: An Anti-Mouse CCR9 Monoclonal Antibody for Immunocytochemistry.

C-C motif chemokine receptor 9 (CCR9) is a G protein-coupled receptor, which is highly expressed in T-lymphocytes and different cancer cells. CCR9 aggravates immune diseases and cancer progression and is considered a biomarker and a therapeutic target of diseases. The development of specific monoclonal antibody (mAbs) for human CCR9 (hCCR9) is required to diagnose and treat immune diseases and cancers. Previously, we established the cell-based immunization and screening (CBIS) method, which does not need purified target proteins. Anti-hCCR9 mAb (clone C9Mab-1; mouse IgG1, kappa) was also developed using the CBIS method. C9Mab-1 is usable for flow cytometry against exogenously and endogenously expressing hCCR9. This study showed that C9Mab-1 and its recombinant antibody (recC9Mab-1) specifically detected exogenous hCCR9 stably overexpressed in Chinese hamster ovary (CHO)-K1 cells and endogenous hCCR9 expressed in a human T-lymphoblastic leukemia cell line MOLT-4 cells through immunocytochemistry. This study provides a new application of C9Mab-1 and recC9Mab-1 in immunocytochemistry.

Purification of Membrane Proteins Overexpressed in Saccharomyces cerevisiae.

Membrane protein (MP) functional and structural characterization requires large quantities of high-purity protein for downstream studies. Barriers to MP characterization include ample overexpression, solubilization, and purification of target proteins while maintaining native activity and structure. These barriers can be overcome by utilizing an efficient purification protocol in a high-yield eukaryotic expression system such as Saccharomyces cerevisiae. S. cerevisiae offers improved protein folding and posttranslational modifications compared to prokaryotic expression systems. This chapter contains practices used to overcome barriers of solubilization and purification using S. cerevisiae that are broadly applicable to diverse membrane associated, and membrane integrated, protein targets.

Modulation of physicochemical properties of magnetic agarose microspheres by hydrolysis-suppressive sequential crosslinking

Separation and purification of target proteins are essential for drug discovery and development. A magnetic separation has been exploited due to its high efficiency. Despite many studies on chemical crosslinking of a magnetic agarose microsphere (MAM), the study on a crosslinking reaction preventing MAM from hydrolytic degradation during the reaction and the effects of the crosslinks on the physicochemical and electrochemical properties of the crosslinked MAM (CL-MAM) has rarely been reported. In our study, CL-MAM was prepared using a sequential crosslinking by which MAM was crosslinked using epichlorohydrin (ECH) in DMSO after crosslinking the MAM by 1,3-dichloro-2-propanol (DCP) for a short time. Such a crosslinking prevented the hydrolytic degradation of DCP-crosslinked MAM ( D -CL-MAM) and DCP and ECH-crosslinked MAM (DE-CL-MAM). MAM had a mean diameter of 110 ± 57.42 µm and its morphology was not affected by the crosslinking. In FT-IR analysis, increase of peak intensity at 1037 cm −1 confirmed newly formed C-O-C bonds in the agarose of CL-MAM. Thermal stability of MAM increased by both DCP crosslinking and subsequent ECH crosslinking of the agarose. While T g of MAM was not shifted by the crosslinking, the onset temperatures of glass transition and thermal decomposition increased by DCP crosslinking and further increased by DCP and ECH-crosslinking. Stiffness (S) and elastic modulus (E) of MAM were enhanced by DCP crosslinking and subsequent ECH crosslinking, indicating a half of ECH formed crosslinks between agarose mainchains while the other half crosslinked adjacent pyranose rings in the agarose mainchain. Permittivity analysis revealed that α relaxation temperature of the agarose film corresponded to T g determined by DSC analysis and the relaxation intensity decreased with increasing crosslink density of the agarose. The higher swelling ratio of DE-CL-MAM compared to D -CL-MAM was attributed to greater S and E values of DE-CL-MAM. The hydrolysis-repressive sequential crosslinking of MAM using DCP and ECH can be a feasible approach to modulate physicochemical and electrochemical properties of MAM. • Hydrolysis-suppressive crosslinkings by DCP and ECH were effective to crosslink MAM. • DCP-crosslinked MAM had enough stability to be crosslinkable by ECH in DMSO. • The sequential crosslinkings did not induce morphological changes of the MAM. • The crosslinking improved thermal stability of MAM, not influencing T g . • Mechanical and swelling properties of crosslinked MAM depended on degree of crosslinks.

PfHRP2 detection using plasmonic optrodes: performance analysis

BackgroundEarly malaria diagnosis and its profiling require the development of new sensing platforms enabling rapid and early analysis of parasites in blood or saliva, aside the widespread rapid diagnostic tests (RDTs).MethodsThis study shows the performance of a cost-effective optical fiber-based solution to target the presence of Plasmodium falciparum histidine-rich protein 2 (PfHRP2). Unclad multimode optical fiber probes are coated with a thin gold film to excite Surface Plasmon Resonance (SPR) yielding high sensitivity to bio-interactions between targets and bioreceptors grafted on the metal surface.ResultsTheir performances are presented in laboratory conditions using PBS spiked with growing concentrations of purified target proteins and within in vitro cultures. Two probe configurations are studied through label-free detection and amplification using secondary antibodies to show the possibility to lower the intrisic limit of detection.ConclusionsAs malaria hits millions of people worldwide, the improvement and multiplexing of this optical fiber technique can be of great interest, especially for a future purpose of using multiple receptors on the fiber surface or several coated-nanoparticles as amplifiers.

Development of a Mucus Gland Bioreactor in Loach Paramisgurnus dabryanus.

Most currently available bioreactors have some defects in the expression, activity, or purification of target protein and peptide molecules, whereas the mucus gland of fish can overcome these defects to become a novel bioreactor for the biopharmaceutical industry. In this study, we have evaluated the practicability of developing a mucus gland bioreactor in loach (Paramisgurnus dabryanus). A transgenic construct pT2-krt8-IFN1 was obtained by subcloning the promoter of zebrafish keratin 8 gene and the type I interferon (IFN1) cDNA of grass carp into the SB transposon. The IFN1 expressed in CIK cells exhibited an antiviral activity against the replication of GCRV873 and activated two genes downstream of JAK-STAT signaling pathway. A transgenic loach line was then generated by microinjection of the pT2-krt8-IFN1 plasmids and in vitro synthesized capped SB11 mRNA. Southern blots indicated that a single copy of IFN1 gene was stably integrated into the genome of transgenic loach. The expression of grass carp IFN1 in transgenic loaches was detected with RT-PCR and Western blots. About 0.0825 µg of grass carp IFN1 was detected in 20 µL mucus from transgenic loaches. At a viral titer of 1 × 103 PFU/mL, plaque numbers on plates containing mucus from transgenic loaches reduced by 18% in comparison with those of the control, indicating that mucus of IFN1-transgenic loaches exhibited an antiviral activity. Thus, we have successfully created a mucus gland bioreactor that has great potential for the production of various proteins and peptides.

A Target-Based In Vivo Test System to Identify Novel Fungicides with Mode of Action in the HOG Pathway.

Resistance management plays a key role in modern plant protection. There is a growing need to identify new fungicide targets and new modes of action. In this context, it is also mandatory to find new compounds acting on successful target locations. For the latter, so-called target-site-specific test systems emerged to search for inhibitors. Most of them are based on in vitro assays, in which interaction between a compound and a purified target protein is demonstrated. Consequently, getting essential information about potentially toxic effects in the living cell or in the whole organism is not possible. Thus, we present a fluorescent-labelled mutant strain ofthe rice blast fungus Magnaporthe oryzae as a rapid tool for fluorescence-based identification and visualization of fungicides in vivowith the mode of action in the highosmolarity glycerol (HOG)-signaling pathway. The HOG pathway represents an excellent target for antifungal agentssuch as the phenylpyrrole fungicides, since almost no relevant resistances have occurred to date, despite 30years of extensive usage of this fungicideclass.