Specific and Fuzzy Interactions Cooperate in Modulating Protein Half-Life.

Protein degradation is critical for maintaining cellular homeostasis. The 20S proteasome is selective for unfolded, extended polypeptide chains without ubiquitin tags. Sequestration of such segments by protein partners, however, may provide a regulatory mechanism.

A Shorter Route to Antibody Binders via Quantitative in vitro Bead-Display Screening and Consensus Analysis.

Affinity panning of large libraries is a powerful tool to identify protein binders. However, panning rounds are followed by the tedious re-screening of the clones obtained to evaluate binders precisely. In a first application of Bead Surface Display (BeSD) we show successful in vitro affinity selections based on flow cytometric analysis that allows fine quantitative discrimination between binders.

Directed evolution of anti-HER2 DARPins by SNAP display reveals stability/function trade-offs in the selection process.

In vitro display technologies have proved to be powerful tools for obtaining high-affinity protein binders. We recently described SNAP display, an entirely in vitro DNA display system that uses the SNAP-tag to link protein with its encoding DNA in water-in-oil emulsions. Here, we apply SNAP display for the affinity maturation of a designed ankyrin repeat proteins (DARPin) that binds to the extracellular domain of HER2 previously isolated by ribosome display.

Handicap-Recover Evolution Leads to a Chemically Versatile, Nucleophile-Permissive Protease.

Mutation of the tobacco etch virus (TEV) protease nucleophile from cysteine to serine causes an approximately ∼10 -fold loss in activity. Ten rounds of directed evolution of the mutant, TEV , overcame the detrimental effects of nucleophile exchange to recover near-wild-type activity in the mutant TEV X. Rather than respecialising TEV to the new nucleophile, all the enzymes along the evolutionary trajectory also retained the ability to use the original cysteine nucleophile.

An experimental framework for improved selection of binding proteins using SNAP display.

Display technologies (e.g. phage and ribosome display) are powerful tools for selecting and evolving protein binders against various target molecules.

In vitro affinity screening of protein and peptide binders by megavalent bead surface display.

The advent of protein display systems has provided access to tailor-made protein binders by directed evolution. We introduce a new in vitro display system, bead surface display (BeSD), in which a gene is mounted on a bead via strong non-covalent (streptavidin/biotin) interactions and the corresponding protein is displayed via a covalent thioether bond on the DNA. In contrast to previous monovalent or low-copy bead display systems, multiple copies of the DNA and the protein or peptide of interest are displayed in defined quantities (up to 10(6) of each), so that flow cytometry can be used to obtain a measure of binding affinity.

A single mutation in the core domain of the lac repressor reduces leakiness.

Background: The lac operon provides cells with the ability to switch from glucose to lactose metabolism precisely when necessary. This metabolic switch is mediated by the lac repressor (LacI), which in the absence of lactose binds to the operator DNA sequence to inhibit transcription. Allosteric rearrangements triggered by binding of the lactose isomer allolactose to the core domain of the repressor impede DNA binding and lift repression.

Flexibility and reactivity in promiscuous enzymes.

Best of both worlds: The interplay of active site reactivity and the dynamic character of proteins allows enzymes to be promiscuous and--sometimes--remarkably efficient at the same time. This review analyses the roles structural flexibility and chemical reactivity play in the catalytic mechanism of selected enzymes.

Promiscuity, stability and cold adaptation of a newly isolated acylaminoacyl peptidase.

We report on the characterisation of a member of the acylaminoacyl peptidase family, the first isolated from bacteria. The enzyme was obtained from the psychrophilic bacterium Sporosarcina psychrophila and shows the typical features of cold adaptation (low T(m), optimal temperature of 40 °C, poor thermal stability). It was also tested for substrate specificity, effect of metals, temperature dependence and structure stability and revealed promiscuous catalytic activity on at least two chemically distinct substrates, with k(cat)/K(m) values for ester hydrolysis and acylamino acids cleavage of 1.

Concepts and tools to exploit the potential of bacterial inclusion bodies in protein science and biotechnology.

Cells have evolved complex and overlapping mechanisms to protect their proteins from aggregation. However, several reasons can cause the failure of such defences, among them mutations, stress conditions and high rates of protein synthesis, all common consequences of heterologous protein production. As a result, in the bacterial cytoplasm several recombinant proteins aggregate as insoluble inclusion bodies.

How disorder influences order and vice versa--mutual effects in fusion proteins containing an intrinsically disordered and a globular protein.

Intrinsically disordered proteins (IDPs) are functional proteins either fully or partly lacking stable secondary and tertiary structure under physiological conditions that are involved in important biological functions, such as regulation and signalling in eukaryotes, prokaryotes and viruses. The function of many IDPs relies upon interactions with partner proteins, often accompanied by conformational changes and disorder-to-order transitions in the unstructured partner. To investigate how disordered and ordered regions interact when fused to one to another within the same protein, we covalently linked the green fluorescent protein to three different, well characterized IDPs and analyzed the conformational properties of the fusion proteins using various biochemical and biophysical approaches.

Evolution of stability in a cold-active enzyme elicits specificity relaxation and highlights substrate-related effects on temperature adaptation.

Molecular aspects of thermal adaptation of proteins were studied by following the co-evolution of temperature dependence, conformational stability, and substrate specificity in a cold-active lipase modified via directed evolution. We found that the evolution of kinetic stability was accompanied by a relaxation in substrate specificity. Moreover, temperature dependence and selectivity turned out to be mutually dependent.

Plasma-induced graft-polymerization of polyethylene glycol acrylate on polypropylene films: chemical characterization and evaluation of the protein adsorption.

This work deals with the optimization of argon plasma-induced graft-polymerization of polyethylene glycol acrylate (PEGA) on polypropylene (PP) films in order to obtain surfaces with a reduced protein adsorption for possible biomedical applications. To this end, we examined the protein adsorption on the treated and untreated surfaces. The graft-polymerization process consisted of four steps: (a) plasma pre-activation of the PP substrates; (b) immersion in a PEGA solution; (c) argon plasma-induced graft-polymerization; (d) washing and drying of the samples.

Components of the E. coli envelope are affected by and can react to protein over-production in the cytoplasm.

Background: Protein over-expression in bacteria is still the easiest, cheapest and therefore preferred way to obtain large amounts of proteins for industrial and laboratory scale preparations. Several studies emphasized the importance of understanding cellular and molecular mechanisms triggered by protein over-production in order to obtain higher yield and better quality of the recombinant product. Almost every step leading to a fully functional polypeptide has been investigated, from mRNA stability to the role of molecular chaperones, from aggregation to bottlenecks in the secretory pathway.

Unscrambling thermal stability and temperature adaptation in evolved variants of a cold-active lipase.

Directed evolution by error-prone PCR was applied to stabilize the cold-active lipase from Pseudomonas fragi (PFL). PFL displays high activity at 10 degrees C, but it is highly unstable even at moderate temperatures. After two rounds of evolution, a variant was generated with a 5-fold increase in half-life at 42 degrees C and a shift of 10 degrees C in the temperature optimum, nevertheless retaining cold-activity.

Fourier transform infrared spectroscopy analysis of the conformational quality of recombinant proteins within inclusion bodies.

The solubility of recombinant proteins produced in bacterial cells is considered a key issue in biotechnology as most overexpressed polypeptides undergo aggregation in inclusion bodies, from which they have to be recovered by solubilization and refolding procedures. Physiological and molecular strategies have been implemented to revert or at least to control aggregation but they often meet only partial success and have to be optimized case by case. Recent studies have shown that proteins embedded in inclusion bodies may retain residual structure and biological function and question the former axiom that solubility and activity are necessarily coupled.

Kinetics of inclusion body formation studied in intact cells by FT-IR spectroscopy.

The aggregation of a recombinant lipase as inclusion bodies (IBs) was studied directly within intact Escherichia coli cells by FT-IR microspectroscopy. Through this approach, it was possible to monitor in real time the different kinetics of IB formation at 37 and 27 degrees C, in excellent agreement with the results of the SDS-PAGE analysis. Furthermore, insights on the residual native-like structure of the expressed protein within IB--both isolated and inside cells--were obtained by the secondary structure analysis of the Amide I band in the IB FT-IR spectra.

Mutations in the "lid" region affect chain length specificity and thermostability of a Pseudomonas fragi lipase.

The cold-adapted Pseudomonas fragi lipase (PFL) displays highest activity on short-chain triglyceride substrates and is rapidly inactivated at moderate temperature. Sequence and structure comparison with homologous lipases endowed with different substrate specificity and stability, pointed to three polar residues in the lid region, that were replaced with the amino acids conserved at equivalent positions in the reference lipases. Substitutions at residues T137 and T138 modified the lipase chain-length preference profile, increasing the relative activity towards C8 substrates.