Selection of TNF-alpha binding affibody molecules using a beta-lactamase protein fragment complementation assay.

Protein fragment complementation assays (PCAs) based on different reporter proteins have been described as powerful tools for monitoring dynamic protein-protein interactions in living cells. The present study describes the construction of a PCA system based on genetic splitting of TEM-1 beta-lactamase for the selection of proteins specifically interacting in the periplasm of Escherichia coli bacterial cells, and its application for the selection of affibody molecules binding human tumour necrosis factor-alpha (TNF-alpha) from a combinatorial library. Vectors encoding individual members of a naïve 10(9) affibody protein library fused to a C-terminal fragment of the beta-lactamase reporter were distributed via phage infection to a culture of cells harbouring a common construct encoding a fusion protein between a non-membrane anchored version of a human TNF-alpha target and the N-terminal segment of the reporter.

Consequences of membrane protein overexpression in Escherichia coli.

Overexpression of membrane proteins is often essential for structural and functional studies, but yields are frequently too low. An understanding of the physiological response to overexpression is needed to improve such yields. Therefore, we analyzed the consequences of overexpression of three different membrane proteins (YidC, YedZ, and LepI) fused to green fluorescent protein (GFP) in the bacterium Escherichia coli and compared this with overexpression of a soluble protein, GST-GFP.

A novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein.

A high throughput method for screening of protein expression is described. By using a flow cytometer, levels of both soluble and precipitated protein can simultaneously be assessed in vivo. Protein fragments were fused to the N-terminus of enhanced GFP and the cell samples were analysed using a flow cytometer.

Fluorescence-microscopy-based image analysis for analyte-dependent particle doublet detection in a single-step immunoagglutination assay.

A novel fluorescence-microscopy-based image analysis method for classification of singlet and doublet latex particles is demonstrated and applied to a particle-based immunoagglutination assay for quantification of biomolecules in microliter-volume bulk samples. The image analysis method, verified by flow cytometric agglutination analysis, is based on a pattern recognition algorithm employing Gaussian-base-function fitting which allows robust identification and counting of singlets, doublets, and higher agglomerates of fluorescent microparticles. The immunoagglutination assay is experimentally modeled by a biotin-streptavidin interaction, with the goal of both theoretically and experimentally investigating the performance of a general immunoagglutination-based assay.

The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures.

The biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, K(d), in the order of 4x10(-14) M. The strength and specificity of the interaction has led it to be one of the most widely used affinity pairs in molecular, immunological, and cellular assays. However, it has previously been impossible to re-use any streptavidin solid support, since the conditions needed to break the interaction with biotin has led to the denaturation of the streptavidin.

Fluorescent detection of beta-lactamase activity in living Escherichia coli cells via esterase supplementation.

The TEM-1 beta-lactamase protein fragment complementation assay was investigated for its applicability in affinity protein-based interaction studies in Escherichia coli, using an affibody-based model system. Results from co-transformation experiments showed that an ampicillin resistant phenotype was specifically associated with cognate affibody-target pairings. Attempts to monitor beta-lactamase complementation in vitro with the fluorescent beta-lactamase substrates CCF2/AM and CCF2 showed that E.

Microbead display of proteins by cell-free expression of anchored DNA.

Gene expression technologies where nucleic acid sequences remain physically linked to their corresponding gene products are important tools for selection and identification of rare variants in large protein libraries. Here, we describe a gene expression system, which combines the potential of bead-based suspension array technology (SAT) with gene expression and clonal identification. Using streptavidin-coated polystyrene micrometer-sized beads as solid supports for anchored PCR products, we have investigated conditions for cell-free expression and bioaffinity technology to provide clonal co-anchoring of corresponding gene products.

Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A.

Factor VIII-specific affibodies were selected from phage displayed libraries constructed by combinatorial mutagenesis of an alpha helical bacterial receptor domain derived from staphylococcal protein A. Bead-immobilized recombinant human factor VIII (rVIII) (80 and 90 kDa chains) protein was used during competitive biopannings in the presence of free 80-kDa chain protein, resulting in the selection of several binders that showed dissociation constants (Kd) in the range 100-200 nM as determined by biosensor analyses. One variant (Z[rVIII:3], 90-kDa chain specific) was further characterized in small-scale affinity chromatography experiments, and showed efficient and selective recovery of biologically active rVIII from Chinese hamster ovary cell supernatant-derived feed stocks.