Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay

Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp). The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y) and P1 (glutamine, Q) within the substrate peptide were confirmed as being the most important specificity determinants. In position P1′, glycine (G), serine (S), cysteine (C), alanine (A) and arginine (R) were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E) is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of proteases and their substrates

Affinity maturation of a TNF-α binding affibodymolecule by Darwinian survival selection

The introduction of different methodologies for construction and screening ofcomplex protein libraries has provided powerful means in protein engineeringfor development of molecules with desired traits. A challenge faced in manysituations is to adapt a given methodology for efficient and rapid identification ofthe most interesting variants present in a library. In the present study, theconcept of Darwinian selection based on a growth advantage for clones havingthe desired trait has been investigated. Using a β-lactamase-based ProteinFragment Complementation Assay (PCA), an affinity maturation of a TNF-αbinding affibody molecule of an initial 2 nM affinity for the target has beenperformed. Initial characterization of the PCA system, based on the affinitydriven reconstitution of β-lactamase activity in the periplasm of cells harbouringa library member showing affinity for a co-expressed target protein, showed thatthe system was responsive to promoter induction level, interaction affinity andapplied selection pressure. Using combinatorial protein engineering principles, a107 library of second generation affibody molecules was constructed andsubjected to selection of improved variants by library growth in liquid culture.The results showed that after a pre-selection step on semi-solid media toeliminate non-binding variants, present in majority, two rounds of selection inliquid culture resulted in an enrichment for binders showing up ten-fold higheraffinity to the TNF-α target than the ancestral variant. Biosensor analysesshowed that the major factor for the improved affinity could be attributed toreduced off-rate constants.Uppdaterad från manuskript till artikel: 20100729QC 2010072

On bacterial formats in protein library technology

Millions of years of evolution have resulted in an immense number of different proteins, which participate in virtually every process within cells and thus are of utmost importance for allknown forms of life. In addition, there are several examples of natural proteins which have found use in applications outside their natural environment, such as the use of enzymes infood industry and washing powders or the use of antibodies in diagnostic, bioseparation or therapeutic applications. To improve the performance of proteins in such applications, anumber of techniques, all collectively referred to as ‘protein engineering’, are performed in thelaboratory.Traditionally, methods involving ‘rational design’, where a few alterations are introduced atspecific protein locations to hopefully result in expected improvements have been applied.However, the use of more recent techniques involving a simultaneous construction of a large number of candidate variants (protein libraries) by various diversification principles, fromwhich rare clones showing enhanced properties can be isolated have contributed greatly to thefield of protein engineering.In the present thesis, different protein traits of biotechnological importance have beenaddressed for improvements by the use of such methods, in which there is a crucial need tomaintain a clonal link between the genotype and the phenotype to allow an identification of protein library members isolated by virtue of their functional properties. In all protein library investigations included in this thesis this coupling has been obtained by Escherichia coli bacterialcell-membrane compartmental confinement.In a first study, a combination of error prone PCR and gene-shuffling was applied to the Tobacco Etch Virus (TEV)-protease gene in order to produce collections from which genesencoding variants showing an enhanced soluble expression of the enzyme frequently used inbiotechnology to cleave fusion proteins were identified. Using Green Fluorescence Protein(GFP)-based cell fluorescence analysis, a clone with a five-fold increase in the yield of solubly produced protein was successfully isolated. In a second study, a novel and different GFPbased selection system, in addition also involving targeted in vivo protein degradation principles,was employed for investigations of the substrate sequence space of the same protease. In two additional studies, a selection system denoted Protein Fragment Complementation Assay(PCA), based on the affinity driven structural complementation of a genetically split β-lactamase enzyme was used to identify variants having desired target protein binding abilities,including both specificity and affinity. Using Darwinian principles concerning clonal growth advantages, affibody binding proteins showing sub-nanomolar dissociation constants to thehuman cytokine TNF-α were isolated. Taken together, these studies have shown that the bacterial format is very well suited for use in various aspects of protein library selection.QC 2010072

Prevalence of hand eczema in an adult Swedish population and the relationship to risk occupation and smoking

Using a postal questionnaire the prevalence of hand eczema was determined in a general population of 11 798 individuals aged 20 - 77 years who were randomly drawn from the population records. The response rate was 78.1%. One-year prevalence of hand eczema among women varied between 1.9% and 10.8%, with the highest figure among those aged 30 - 39 years. The corresponding figures for men were 2.3% and 5.6%, with the highest figure among those aged 20 - 29 years. Lifetime prevalence varied between 5.7% and 16.7% among women and between 5.2% and 9.5% among men. Using multiple logistic regression analysis female sex (OR = 1.91, 95% CI 1.47 - 2.47) and smoking (OR = 1.35, 95% CI 1.04 - 1.75) were independent risk factors for reporting 1-year prevalence of hand eczema, whereas age (OR = 0.99, 95% CI 0.97 - 0.99) was inversely related to the 1-year prevalence of hand eczema. Aggregated risk occupation or categorized occupation such as medical and nursing work, production or service were not significantly associated with 1- year prevalence of hand eczema

Prevalence of self-reported eczema in relation to living environment, socio-economic status and respiratory symptoms assessed in a questionnaire study

BACKGROUND: Potential links between eczema and obstructive pulmonary diseases have been postulated. Previously we have reported the prevalence of upper and lower respiratory diseases and the relation to environmental and socio-economic factors in a randomly selected adult population in southern Sweden using a postal questionnaire.In the present study we wanted to analyse the prevalence of eczema and its relation to socio-economic status, heredity factors and environmental factors in an adult population. METHODS: Self-reported eczema, upper and lower respiratory symptoms, asthma and Chronic Bronchitis Emphysema (CBE) were examined in 12,071 adults, aged 20-59 years, living in southern Sweden by using a postal questionnaire. There were comparable numbers of males and females in all age groups.Multiple logistic regression analysis (forward conditional) was applied to estimate the association between the proposed risk factors (heredity, self-reported asthma and CBE, nasal symptoms, socio-