Gerhard Stadlmayr

University of Natural Resources and Life Science Vienna, Wien, Vienna, Austria

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Publications (14)55.24 Total impact

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    ABSTRACT: For most therapeutic proteins, a long serum half-life is desired. Studies have shown that decreased antigen binding at acidic pH can increase serum half-life. In this study we aimed to investigate whether pH-dependent binding sites can be introduced into antigen binding crystallizable fragments of immunoglobulin G1 (Fcab). The C-terminal structural loops of an Fcab were engineered for reduced binding to the extracellular domain of human epidermal growth factor receptor 2 (Her2-ECD) at pH 6 compared to pH 7.4. A yeast-displayed Fcab-library was alternately selected for binding at pH 7.4 and non-binding at pH 6.0. Selected Fcab variants showed clear pH-dependent binding to soluble Her2-ECD (decrease in affinity at pH 6.0 compared to pH 7.4) when displayed on yeast. Additionally, some solubly expressed variants exhibited pH-dependent interactions with Her2-positive cells whereas their conformational and thermal stability was pH-independent. Interestingly, two of the three Fcabs did not contain a single histidine mutation but all of them contained variations next to histidines that already occurred in loops of the lead Fcab. The study demonstrates that yeast surface display is a valuable tool for directed evolution of pH-dependent binding sites in proteins.
    Biotechnology Journal 06/2014; · 3.71 Impact Factor
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    ABSTRACT: Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules.
    Biochimica et Biophysica Acta 05/2014; · 4.66 Impact Factor
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    ABSTRACT: IgM antibodies are an important player of the human's innate defense mechanisms and increasingly have gained interest as therapeutics. Although the expression of IgM antibodies in mammalian cell culture is established, this approach remains costly and alternative methods have not been developed yet. Plants have a proven record for the production of therapeutically relevant recombinant proteins. However, whether they are able to express proteins like IgM antibodies, which range among the most complex human proteins, remains unknown so far. Here we report the in planta generation of the functionally active monoclonal antitumor IgM PAT-SM6 (SM6). SM6 efficiently accumulates in plant leaves and assembles correctly into heterooligomers (pentamers and hexamers). Detailed glycosylation analysis exhibited complex and oligomannosidic N-glycans in a site-specific manner on human-serum IgM and on plant- and human-cell-line-produced SM6. Moreover, extensive in planta glycoengineering allowed the generation of SM6 decorated with sialylated human-type oligosaccharides, comparable to plasma-derived IgM. A glycosylated model of pentameric IgM exhibits different accessibility of the glycosylation sites, explaining site-specific glycosylation. Biochemical and biophysical properties and importantly biological activities of plant-derived SM6 glycoforms are comparable to the human-cell-derived counterparts. The in planta generation of one of the most complex human proteins opens new pathways toward the production of difficult-to-express proteins for pharmaceutical applications. Moreover, the generation of IgMs with a controlled glycosylation pattern allows the study of the so far unknown contribution of sugar moieties to the function of IgMs.
    Proceedings of the National Academy of Sciences 03/2014; · 9.81 Impact Factor
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    ABSTRACT: Chinese hamster ovary (CHO) cells are currently the workhorse of the biopharmaceutical industry. However, yeasts such as Pichia pastoris are about to enter this field. To compare their capability for recombinant protein secretion, P. pastoris strains and CHO cell lines producing human serum albumin (HSA) and the 3D6 single chain Fv-Fc anti-HIV-1 antibody (3D6scFv-Fc) were cultivated in comparable fed batch processes. In P. pastoris, the mean biomass-specific secretion rate (qP ) was 40-fold lower for 3D6scFv-Fc compared to HSA. On the contrary, qP was similar for both proteins in CHO cells. When comparing both organisms, the mean qP of the CHO cell lines was 1011-fold higher for 3D6scFv-Fc and 26-fold higher for HSA. Due to the low qP of the 3D6scFv-Fc producing strain, the space-time yield (STY) was 9.6-fold lower for P. pastoris. In contrast, the STY of the HSA producer was 9.2-fold higher compared to CHO cells because of the shorter process time and higher biomass density. The results indicate that the protein secretion machinery of P. pastoris is much less efficient and the secretion rate strongly depends on the complexity of the recombinant protein. However, process efficiency of the yeast system allows higher STYs for less complex proteins.
    Biotechnology Journal 01/2014; · 3.71 Impact Factor
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    ABSTRACT: Figure optionsDownload full-size imageDownload high-quality image (117 K)Download as PowerPoint slide
    Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 01/2014; · 3.73 Impact Factor
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    ABSTRACT: Antigen-binding Fc fragments (Fcab) are generated by engineering the C-terminal loop regions in the CH3 domain of human immunoglobulin G class 1-crystallizable fragment (IgG1-Fc). For an optimum library design with high percentage of well-folded clones for efficient binder selection, information about the correlation between primary structure and stability is needed. Here, we present a rapid method that allows determination of the overall stability of whole libraries of IgG1-Fc on the surface of yeast by flow cytometry. Libraries of IgG1-Fc mutants with distinct regions in AB-, CD- and EF-loops of the CH3 domains randomized or carrying therein insertions of five additional residues were constructed, incubated at increasing temperatures and probed for residual binding of generic Fc ligands. Calculated temperatures of half-maximal irreversible denaturation of the libraries gave a clear hierarchy of tolerance to randomization of distinct loop positions. Experimental data were evaluated by a computational approach and are discussed with respect to the structure of IgG1-Fc and variation in sequence and length of these loops in homologous Fc proteins. Generally, the described method allows for quick assessment of the effects of randomization of distinct regions on the foldability and stability of a yeast-displayed protein library.
    Protein Engineering Design and Selection 09/2013; · 2.59 Impact Factor
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    ABSTRACT: An Fcab (Fc antigen binding) is a crystallizable fragment of IgG having C-terminal structural loops of CH3 domains engineered for antigen binding. Since introduction of novel binding sites might impair the immunoglobulin fold, repairing strategies are needed for improving the biophysical properties of promising binders without decreasing affinity to the antigen. Here, a directed evolution protocol was developed and applied for stabilization of a Her2/neu-binding Fcab. Distinct loop regions of the parental binder were softly randomized by parsimonious mutagenesis, followed by heat incubation of the yeast displayed protein library and selection for retained antigen binding. Selected Fcabs were expressed solubly in Pichia pastoris and human embryonic kidney 293 cells and characterized. Fcab clones that retained their affinity to Her2/neu but exhibited a significantly increased conformational stability and resistance to aggregation could be evolved. Moreover, we demonstrate that simultaneous selection for binding to the antigen and to structurally specific ligands (FcγRI and an antibody directed against the CH2 domain) yields even more stable Fcabs. To sum up, this study presents a very potent and generally applicable method for improving the fold and stability of antibodies, antibody fragments and alternative binding scaffolds.
    Protein Engineering Design and Selection 12/2012; · 2.59 Impact Factor
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    ABSTRACT: Genetic fusion of two proteins frequently induces beneficial effects to the proteins, such as increased solubility, besides the combination of two protein functions. Here, we study the effects of the bacterial surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a on the folding of a C-terminally fused enhanced green fluorescent protein (EGFP) moiety. Although GFPs are generally unable to adopt a functional confirmation in the bacterial periplasm of Escherichia coli cells, we observed periplasmic fluorescence from a chimera of a 150-amino-acid N-terminal truncation of SgsE and EGFP. Based on this finding, unfolding and refolding kinetics of different S-layer-EGFP chimeras, a maltose binding protein-EGFP chimera, and sole EGFP were monitored using green fluorescence as indicator for the folded protein state. Calculated apparent rate constants for unfolding and refolding indicated different folding pathways for EGFP depending on the fusion partner used, and a clearly stabilizing effect was observed for the SgsE_C fusion moiety. Thermal stability, as determined by differential scanning calorimetry, and unfolding equilibria were found to be independent of the fused partner. We conclude that the stabilizing effect SgsE_C exerts on EGFP is due to a reduction of degrees of freedom for folding of EGFP in the fused state.
    Journal of Microbiology and Biotechnology 09/2012; 22(9):1271-8. · 1.40 Impact Factor
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    ABSTRACT: One of the most important but still poorly understood issues in protein chemistry is the relationship between sequence and stability of proteins. Here, we present a method for analyzing the influence of each individual residue on the foldability and stability of an entire protein. A randomly mutated library of the crystallizable fragment of human immunoglobulin G class 1 (IgG1-Fc) was expressed on the surface of yeast, followed by heat incubation at 79°C and selection of stable variants that still bound to structurally specific ligands. High throughput sequencing allowed comparison of the mutation rate between the starting and selected library pools, enabling the generation of a stability landscape for the entire CH3 domain of human IgG1 at single residue resolution. Its quality was analyzed with respect to (i) the structure of IgG1-Fc, (ii) evolutionarily conserved positions and (iii) in silico calculations of the energy of unfolding of all variants in comparison with the wild-type protein. In addition, this new experimental approach allowed the assignment of functional epitopes of structurally specific ligands used for selection [Fc γ-receptor I (CD64) and anti-human CH2 domain antibody] to distinct binding regions in the CH2 domain.
    Journal of Molecular Biology 07/2012; 423(3):397-412. · 3.91 Impact Factor
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    ABSTRACT: We have constructed IgG1-Fc scaffolds with increased thermal stability by directed evolution and yeast surface display. As a basis a new selection strategy that allowed the application of yeast surface display for screening of stabilizing mutations in proteins of already high intrinsic thermal stability and T(m)-values up to 85°C was developed. Besides library construction by error prone PCR, strong heat stress at 79°C for 10min and screening for well-folded proteins by FACS, sorting rounds had to include an efficient plasmid DNA isolation step for amplification and further transfection. We describe the successful application of this experimental setup for selection of 17 single, double and triple IgG1-Fc variants of increased thermal stability after four selection rounds. The recombinantly produced homodimeric proteins showed a wild-type-like elution profile in size exclusion chromatography as well as content of secondary structures. Moreover, the kinetics of binding of FcRn, CD16a and Protein A to the engineered Fc-molecules was very similar to the wild-type protein. These data clearly demonstrate the importance and efficacy of the presented strategy for selection of stabilizing mutations in proteins of high intrinsic stability within reasonable time.
    Biochimica et Biophysica Acta 01/2012; 1824(4):542-9. · 4.66 Impact Factor
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    ABSTRACT: Recently, it has been demonstrated that loops of the crystallizable fragment of IgG1 (IgG1-Fc) can be engineered to form antigen-binding sites. In this work C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc have been functionalized to form integrin-binding sites in order to probe the effect of engineering on structural integrity and thermal stability of IgG1-Fc as well as on binding to the ligands Protein A, CD16 and FcRn, respectively. The peptide sequence GCRGDCL--a disulfide-bridged cyclic heptapeptide that confers binding to human αvβ3 integrin was introduced into AB, CD and/or EF loops and single and double mutants were heterologously expressed in Pichia pastoris. Integrin binding of engineered IgG-Fc was tested using both binding to coated αvβ3 integrin in ELISA or to αvβ3-expressing K562 cells in FACS analysis. Additionally, blocking of αvβ3-mediated cell adhesion to vitronectin was investigated. The data presented in this report demonstrate that bioactive integrin-binding peptide(s) can be grafted on the C-terminal loops of IgG-Fc without impairing binding to effector molecules. Observed differences between the investigated variants in structural stability and integrin binding are discussed with respect to the known structure of IgG-Fc and its structural loops.
    Journal of Biotechnology 09/2011; 155(2):193-202. · 3.18 Impact Factor
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    ABSTRACT: The number of available promoters for the protein production host Pichia pastoris is limited, and in most applications comprises the methanol inducible alcohol oxidase 1 (AOX1) promoter and the constitutive glyceraldehyde phosphate dehydrogenase (GAP) promoter. To close this gap, we identified 24 novel potential regulatory sequences and tested their applicability to drive the expression of both intracellular as well as secretory heterologous proteins. While more than 80% of the promoters derived from microarray data mining showed activity on all common carbon sources used for P. pastoris, the success rate of rationally selected promoters was lower. Many of the newly identified promoters showed a growth rate dependent behaviour, for example three ribosomal promoters as well as the promoters of two chaperones were much more active at early growth phase as compared to later phases. Fed batch cultivation of selected promoters expressing human serum albumin further strengthened the correlation of promoter activity (determined by HSA transcript levels) to specific growth rate. The promoter of the thiamine biosynthesis gene P(THI11) did not only show high transcript levels at low specific growth rate, but also exhibited interesting regulatory properties dependent on the availability of thiamine in the growth medium.
    Journal of Biotechnology 10/2010; 150(4):519-29. · 3.18 Impact Factor
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    ABSTRACT: A method combining fluorescence activated cell sorting (FACS) and DNA microarray assisted clone identification was developed and termed Genome-Scale Analysis of Library Sorting (GALibSo). Genes enhancing the production of secreted heterologous proteins in Pichia pastoris were identified out of a cDNA library by cell surface display and FACS. The trends of gene enrichment during consecutive FACS rounds were monitored by DNA microarrays. In a case study a P. pastoris cDNA library was co-expressed in a strain secreting the Fab fragment of a monoclonal antibody against human immunodeficiency virus type 1 as a model protein. Three genes were identified, increasing the relative expression level of the surface-displayed model protein up to 45%. While one of these genes had a positive effect on three out of four tested proteins, the product specific effect of the other two suggested that the effects of the co-expressed secretion enhancing factors are partly dependent on the protein to be produced. The microarray based monitoring of the enrichment of genes causing enhanced protein secretory capacity led to novel insights into the limitation of protein secretion.
    Biotechnology and Bioengineering 10/2009; 105(3):543-55. · 4.16 Impact Factor
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    ABSTRACT: Efficient production of heterologous proteins with yeasts and other eukaryotic hosts is often hampered by inefficient secretion of the product. Limitation of protein secretion has been attributed to a low folding rate, and a rational solution is the overexpression of proteins supporting folding, like protein disulfide isomerase (Pdi), or the unfolded protein response transcription factor Hac1. Assuming that other protein factors which are not directly involved in protein folding may also support secretion of heterologous proteins, we set out to analyze the differential transcriptome of a Pichia pastoris strain overexpressing human trypsinogen versus that of a nonexpressing strain. Five hundred twenty-four genes were identified to be significantly regulated. Excluding those genes with totally divergent functions (like, e.g., core metabolism), we reduced this number to 13 genes which were upregulated in the expression strain having potential function in the secretion machinery and in stress regulation. The respective Saccharomyces cerevisiae homologs of these genes, including the previously characterized secretion helpers PDI1, ERO1, SSO2, KAR2/BiP, and HAC1 as positive controls, were cloned and overexpressed in a P. pastoris strain expressing a human antibody Fab fragment. All genes except one showed a positive effect on Fab fragment secretion, as did the controls. Six out of these novel secretion helper factors, more precisely Bfr2 and Bmh2 (involved in protein transport), the chaperones Ssa4 and Sse1, the vacuolar ATPase subunit Cup5, and Kin2 (a protein kinase connected to exocytosis), proved their benefits for practical application in laboratory-scale production processes by increasing both specific production rates and the volumetric productivity of an antibody fragment up to 2.5-fold in fed-batch fermentations of P. pastoris.
    Applied and Environmental Microbiology 11/2007; 73(20):6499-507. · 3.95 Impact Factor