Andreas Christmann

Gene Islands Integrated into tRNA(Gly) Genes Confer Genome Diversity on a Pseudomonas aeruginosa Clone

Intraclonal genome diversity of Pseudomonas aeruginosa was studied in one of the most diverse mosaic regions of the P. aeruginosa chromosome. The ca. 110-kb large hypervariable region located near the lipH gene in two members of the predominant P. aeruginosa clone C, strain C and strain SG17M, was sequenced. In both strains the region consists of an individual strain-specific gene island of 111 (strain C) or 106 (SG17M) open reading frames (ORFs) and of a 7-kb stretch of clone C-specific sequence of 9 ORFs. The gene islands are integrated into conserved tRNA(Gly) genes and have a bipartite structure. The first part adjacent to the tRNA gene consists of strain-specific ORFs encoding metabolic functions and transporters, the majority of which have homologs of known function in other eubacteria, such as hemophores, cytochrome c biosynthesis, or mercury resistance. The second part is made up mostly of ORFs of yet-unknown function. Forty-seven of these ORFs are mutual homologs with a pairwise amino acid sequence identity of 35 to 88% and are arranged in the same order in the two gene islands. We hypothesize that this novel type of gene island derives from mobile elements which, upon integration, endow the recipient with strain-specific metabolic properties, thus possibly conferring on it a selective advantage in its specific habitat.

Sequence requirements of the GPNG beta-turn of the Ecballium elaterium trypsin inhibitor II explored by combinatorial library screening.

The Ecballium elaterium trypsin inhibitor II (EETI-II) contains 28 amino acids and three disulfides forming a cystine knot. Reduced EETI-II refolds spontaneously and quantitatively in vitro and regains its native structure. Due to its high propensity to form a reverse turn, the GPNG sequence of segment 22–25 comprising a β-turn in native EETI-II is a possible candidate for a folding initiation site. We generated a molecular repertoire of EETI-II variants with variegated 22–25 tetrapeptide sequences and presented these proteins on the outer membrane ofEscherichia coli cells via fusion to the Igaβautotransporter. Functional trypsin-binding variants were selected by combination of magnetic and fluorescence-activated cell sorting. At least 1–5% of all possible tetrapeptide sequences were compatible with formation of the correct three disulfides. Occurrence of amino acid residues in functional variants is positively correlated with their propensity to be generally found in β-turns. The folding pathway of two selected variants, EETI-βNEDE and EETI-βTNNK, was found to be indistinguishable from EETI-II and occurs through formation of a stable 2-disulfide intermediate. Substantial amounts of misfolded byproducts, however, were obtained upon refolding of these variants corroborating the importance of the wild type EETI-II GPNG sequence to direct quantitative formation of the cystine knot architecture.

Display of Passenger Proteins on the Surface of Escherichia coli K-12 by the Enterohemorrhagic E. coli Intimin EaeA

Intimins are members of a family of bacterial adhesins from pathogenic Escherichia coli which specifically interact with diverse eukaryotic cell surface receptors. The EaeA intimin from enterohemorrhagic E. coli O157:H7 contains an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passenger domains across the bacterial cell envelope. We investigated whether truncated EaeA intimin lacking two carboxy-terminal domains could be used as a translocator for heterologous passenger proteins. We found that a variant of the trypsin inhibitor Ecballium elaterium trypsin inhibitor II (EETI-II), interleukin 4, and the Bence-Jones protein REI(v) were displayed on the surface of E. coli K-12 via fusion to truncated intimin. Fusion protein net accumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for translational readthrough at an amber codon residing within the truncated eaeA gene. Intimin-mediated adhesion of the bacterial cells to eukaryotic target cells could be mimicked by surface display of a short fibrinogen receptor binding peptide containing an arginine-glycine-aspartic acid sequence motif, which promoted binding of E. coli K-12 to human platelets. Cells displaying a particular epitope sequence fused to truncated intimin could be enriched 200,000-fold by immunofluorescence staining and fluorescence-activated cell sorting in three sorting rounds. These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation of various passenger proteins through the cytoplasmic and outer membranes of E. coli and their exposure on the cell surface. Intimin display may prove a useful tool for future protein translocation studies with interesting biological and biotechnological ramifications.

Epitope mapping and affinity purification of monospecific antibodies by Escherichia coli cell surface display of gene-derived random peptide libraries

We report a method for the precise mapping of linear epitopes by presenting a peptide library on the surface of Escherichia coli cells. A random library of gene fragments derived from the classical swine fever virus (CSFV) envelope protein E(rns) was generated by DNAse I cleavage and cloned into a specially designed bacterial surface display vector. A carboxyterminally truncated intimin, an adhesin from enteropathogenic E. coli, serves as a carrier protein to present foreign peptides on the surface of E. coli K12 cells. Epitope-presenting cells were isolated by immunofluorescence staining of the bacterial cell population with monoclonal anti-E(rns) antibodies followed by fluorescence-activated cell sorting (FACS). Nucleotide sequence analysis of the coding sequence for the cloned target gene fragments of a few FACS-positive clones allowed the identification of the respective epitope sequence. A major linear antigenic determinant of the E(rns) protein could be identified by epitope mapping with a polyclonal anti-E(rns) serum. Furthermore, the high-density surface display of intimin-peptide fusions allowed us to use epitope-presenting bacteria directly as whole cell adsorbants for affinity purification of monospecific antibodies. Monospecific antibodies directed against the carboxyterminal fragment of E(rns) were isolated and used for immunostaining of transfected BHK-21 cells to validate the transient expression of E(rns). This demonstrates that gene-fragment libraries displayed on E. coli cells as fusion proteins with intimin are useful tools for rapid mapping of linear epitopes recognized by monoclonal antibodies (MAbs) and polyclonal sera and for the affinity purification of monospecific antibodies by adsorption to the E. coli surface exposed antigenic peptide.

Combinatorial Optimization of Cystine-Knot Peptides towards High-Affinity Inhibitors of Human Matriptase-1

Cystine-knot miniproteins define a class of bioactive molecules with several thousand natural members. Their eponymous motif comprises a rigid structured core formed by six disulfide-connected cysteine residues, which accounts for its exceptional stability towards thermic or proteolytic degradation. Since they display a remarkable sequence tolerance within their disulfide-connected loops, these molecules are considered promising frameworks for peptide-based pharmaceuticals. Natural open-chain cystine-knot trypsin inhibitors of the MCoTI (Momordica cochinchinensis trypsin inhibitor) and SOTI (Spinacia oleracea trypsin inhibitor) families served as starting points for the generation of inhibitors of matriptase-1, a type II transmembrane serine protease with possible clinical relevance in cancer and arthritic therapy. Yeast surface-displayed libraries of miniproteins were used to select unique and potent matriptase-1 inhibitors. To this end, a knowledge-based library design was applied that makes use of detailed information on binding and folding behavior of cystine-knot peptides. Five inhibitor variants, four of the MCoTI family and one of the SOTI family, were identified, chemically synthesized and oxidatively folded towards the bioactive conformation. Enzyme assays revealed inhibition constants in the low nanomolar range for all candidates. One subnanomolar binder (Ki = 0.83 nM) with an inverted selectivity towards trypsin and matriptase-1 was identified.

Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.

In vivo enzyme immobilization by inclusion body display.

ABSTRACT A novel strategy for in vivo immobilization of enzymes on the surfaces of inclusion bodies has been established. It relies on expression in Escherichia coli of the polyhydroxybutyrate synthase PhaC from Cupriavidus necator, which carries at its amino terminus an engineered negatively charged α-helical coil (Ecoil) and forms inclusion bodies upon high-level expression. Coexpression in the same cell of galactose oxidase (GOase) from Fusarium spp. carrying a carboxy-terminal positively charged coil (lysine-rich coil [Kcoil]) sequence results in heterodimeric coiled-coil formation in vivo and in the capture of the enzyme in active form on the surface of the inclusion body particle. These round-shaped enzyme-decorated microparticles, with sizes of approximately 0.7 μm, can be isolated from lysed cells simply by centrifugation. The cost-effective one-step generation and isolation of enzymes immobilized on inclusion body particles may become useful for various applications in bioprocessing and biotransformation.

Bacteria Displaying Interleukin‐4 Mutants Stimulate Mammalian Cells and Reflect the Biological Activities of Variant Soluble Cytokines

We describe a novel procedure that allows the rapid determination of cytokine activity on cells that express their cognate receptor. The four‐helix bundle cytokine interleukin‐4 (IL‐4) was inducibly expressed as a fusion with the E. coli outer‐membrane protein intimin, such that IL‐4 was presented on the surfaces of the bacteria. Expression and accessibility of the cytokine on the cell exteriors were monitored by Western blotting and fluorescence microscopy, making use of two epitopes flanking the IL‐4 component of the fusion protein. To demonstrate the biological activity of the immobilized cytokine, a Ba/F3‐derived cell line stably transfected with both the bipartite human IL‐4 receptor and an IL‐4‐specific luciferase reporter gene construct was employed. Bacterial cells displaying interleukin‐4 elicited a specific, dose‐dependent response in the reporter cells. Two variants of IL‐4 with previously characterized (partial) antagonistic properties were also expressed as membrane‐bound fusion proteins and were tested for their activity in the immobilized state. In comparison with bacteria displaying wild‐type IL‐4, E. coli clones presenting variants IL‐4 Y124G and Y124D showed diminished or abolished activity, respectively, on murine reporter cells. The relative signaling potencies of the immobilized IL‐4 variants thus closely mirror the agonistic properties of the corresponding soluble cytokines. This approach should be generally applicable for the mutational analysis of numerous signal mediators that trigger cellular responses through dimerization of transmembrane receptors.

Directed Evolution of a Bond-Forming Enzyme: Ultrahigh-Throughput Screening of Microbial Transglutaminase Using Yeast Surface Display

Microbial transglutaminase from Streptomyces mobaraensis (mTG) has emerged as a useful biotechnological tool due to its ability to crosslink a side chain of glutamine and primary amines. To date, the substrate specificity of mTG is not fully understood, which poses an obvious challenge when mTG is used to address novel targets. To that end, a viable strategy providing an access to tailor-made transglutaminases is required. This work reports an ultrahigh-throughput screening approach based on yeast surface display and fluorescence-activated cell sorting (FACS) that enabled the evolution of microbial transglutaminase towards enhanced activity. Five rounds of FACS screening followed by recombinant expression of the most potent variants in E. coli yielded variants that possessed, compared to the wild type enzyme, improved enzymatic performance and labeling behavior upon conjugation with an engineered therapeutic anti-HER2 antibody. This robust and generally applicable platform enables tailoring of the catalytic efficiency of mTG.