The biotin-streptavidin interaction can be reversibly broken using water at elevated temperaturesThe biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, K d , in the order of 4610 214 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. Here, we show that a short incubation in nonionic aqueous solutions at temperatures above 707C can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-use of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid phase applications in fields, such as microfluidics and nanotechnology. IntroductionThe strong interaction between avidin and biotin was discovered as early as 1941 [1]. Avidin is a protein commonly purified from chicken egg white while biotin is a vitamin found in all cells. Streptavidin, a bacterial homologous protein to avidin, isolated from the actinobacterium Streptomyces avidinii, is more frequently used than avidin and is commercially available also in a number of engineered forms. The structure of the biotin-streptavidin complex has been described by several groups [2,3], showing a b-barrel structure of streptavidin binding biotin into its interior. The binding between avidin/streptavidin and biotin has long been regarded as the strongest, noncovalent, biological interaction known, having a dissociation constant, K d , in the order of 4610 214 M [4]. The bond forms very rapidly and is stable in wide ranges of pH and temperature [1,5].The strong interaction has led to a large number of research and diagnostic applications using avidin-biotin or streptavidin-biotin technology. The strength and reliability of the interaction underlie its importance in biotechnology, but the interaction is also a model for high-affinity receptor ligand binding. In most assays, streptavidin is coupled to a solid phase, such as a magnetic bead, a microtiter plate, or a biosensor chip, while biotin is coupled to the moiety of interest, often a nucleic acid, protein, or antibody. However, harsh conditions, such as formamide treatment combined with high temperatures, have been required to separate biotin from streptavidin, resulting in not only denatured streptavidin molecules [5] but also in limitations of downstream applications due to deterioration ...

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. Proteomes of total lysates, purified aggregates, and cytoplasmic membranes were analyzed by one-and two-dimensional gel electrophoresis and mass spectrometry complemented with flow cytometry, microscopy, Western blotting, and pulse labeling experiments. Composition and accumulation levels of protein complexes in the cytoplasmic membrane were analyzed with improved two-dimensional blue native PAGE. Overexpression of the three membrane proteins, but not soluble GST-GFP, resulted in accumulation of cytoplasmic aggregates containing the overexpressed proteins, chaperones (DnaK/J and GroEL/ S), and soluble proteases (HslUV and ClpXP) as well as many precursors of periplasmic and outer membrane proteins. This was consistent with lowered accumulation levels of secreted proteins in the three membrane protein overexpressors and is likely to be a direct consequence of saturation of the cytoplasmic membrane protein translocation machinery. Importantly accumulation levels of respiratory chain complexes in the cytoplasmic membrane were strongly reduced. Induction of the acetate-phosphotransacetylase pathway for ATP production and a downregulated tricarboxylic acid cycle indicated the activation of the Arc two-component system, which mediates adaptive responses to changing respiratory states. This study provides a basis for designing rational strategies to improve yields of membrane protein overexpression in E. coli.

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Factor VIII-specific affibodies were selected from phage displayed libraries constructed by combinatorial mutagenesis of an a 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 (K d ) 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. The purity of the enriched rVIII was comparable with rVIII material purified by immunoaffinity chromatography using a 90-kDa chain-specific monoclonal antibody. Interestingly, epitope mapping showed that the monoclonal antibody and the affibody ligand competed for the same or at least overlapping epitopes on rVIII. In addition, the Z rVIII:3 variant was produced by peptide synthesis with a C-terminal cysteine to enable directed coupling to solid supports. This 59-residue protein was analyzed by circular dichroism and showed a secondary structure content similar to that of the parental Z domain used as scaffold. In biosensor studies, the synthetic affibody was immobilized recruiting the C-terminal cysteine residue, and demonstrated to bind both recombinantly produced and plasmaderived factor VIII. From a secondary library, constructed by re-randomization of relevant positions identified after alignment of the first-generation variants, a panel of affinityimproved second-generation affibodies were selected of which one clone showed a dissociation constant (K d ) for rVIII of 5 nm. Several of these variants also showed higher apparent binding efficiencies towards rVIII when analyzed as immobilized ligands in biosensor experiments. Taken together, the results suggest that affibody ligands produced by bacterial or synthetic routes could be of interest as an alternative to monoclonal antibodies in purification processes or as diagnostic or monitoring tools.

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