A universal approach for promoter strength evaluation supported by the web-based tool PromCal.

In genetic engineering, gene expression is often modulated by replacements in promoter regions. Any deliberate intervention into the regulatory elements requires a subsequent evaluation based on analysis of reporter proteins. We have developed a new and rapid approach for characterization of promoter activity in which promoter strengths are determined by antibiotic resistance level. Values are expressed in comparison with those obtained from the reference promoter using the kanamycin resistance (aminoglycoside 3'-phosphotransferase) gene as a reporter. The new assay vector pSB1K0prom enables straightforward cloning of promoters or their subparts; therefore, mutations in different elements of the promoter region are easily introduced and analyzed. A series of promoters can be examined in parallel because no protein analysis is required other than determination of bacterial growth rates in the presence of increasing kanamycin concentrations. An internet application called PromCal for evaluation of experimental data has also been developed and is freely accessible at http://web.fkkt.uni-lj.si/biokemija/nskrlj/tools/PromCal.php.

A versatile bacterial expression vector based on the synthetic biology plasmid pSB1.

We have developed an Escherichia coli expression vector that is particularly useful for construction and production of fusion proteins. Based on the synthetic biology pSB1C3 platform, the resulting vector offers a combination of useful features: the strong T7 promoter combined with lac operator, OmpA signal sequence, a selection of cloning sites located at convenient positions and a 3'-terminal His-10 tag. Each of these regions is flanked by a restriction site that allows for easy vector modification, including removal of the signal sequence without perturbation of the reading frame. All the elements were assembled by stepwise addition of three cassettes for which the design was made de novo. To prove the efficiency of the new vector, named pMD204, we successfully produced a cysteine proteinase inhibitor variant in the periplasm and in the cytoplasm of E. coli, in both cases as a soluble and active protein.

Recombinant human SMOCs produced by in vitro refolding: calcium-binding properties and interactions with serum proteins.

Secreted modular calcium-binding (SMOC) proteins are little known members of the BM-40 family of matricellular proteins. SMOC-1 is localized in basement membranes, while SMOC-2 exhibits pro-angiogenic properties and stimulates cell cycle progression via integrin-linked kinase. In this work we have expressed recombinant human SMOCs in inclusion bodies in Escherichia coli. Soluble proteins were prepared by in vitro refolding with a final yield of approximately 3mg of purified SMOCs per liter of bacterial culture. The folding state of the products and their ability to reversibly bind calcium ions were verified by CD spectroscopy. The EF hands of the refolded SMOCs were both functional, one had high affinity for calcium ions (K(d) values in the 0.7-1 microM range), while the other had lower affinity (K(d) values 20-25 microM). The proteins were also examined for their ability to bind blood serum proteins. Three of the bands specifically retained on SMOC-Sepharose were identified as C-reactive protein, an acute phase protein from the pentraxin family, the basement membrane and elastic fiber-associated fibulin-1, and vitronectin, which is involved in cell adhesion, migration and proliferation and binds numerous extracellular and membrane proteins, including integrins. The interactions were additionally confirmed in solution using purified individual proteins by the method of biotin label transfer from one interacting partner to the other. Their identification is among the first pieces of information about the action of the SMOCs on molecular level and opens new possibilities for future research aimed towards elucidating the physiological roles of these versatile proteins.

New engineered antibodies against prions.

A number of recently developed and approved therapeutic agents based on highly specific and potent antibodies have shown the potential of antibody therapy. As the next step, antibody-based therapeutics will be bioengineered in a way that they not only bind pathogenic targets but also address other issues, including drug targeting and delivery. For antibodies that are expected to act within brain tissue, like those that are directed against the pathogenic prion protein isoform, one of the major obstacles is the blood-brain barrier which prevents efficient transfer of the antibody, even of the engineered single-chain variants. We recently demonstrated that a specific prion-specific antibody construct which was injected into the murine tail vein can be efficiently transported into brain tissue. The novelty of the work was in that the cell penetrating peptide was used as a linker connecting both specificity-determining domains of the antibody peptide, thus eliminating the need for the standard flexible linker, composed of an arrangement of three consecutive (Gly 4Ser) repeats. This paves the road toward improved bioengineered antibody variants that target brain antigens.

Recombinant single-chain antibody with the Trojan peptide penetratin positioned in the linker region enables cargo transfer across the blood-brain barrier.

Delivery of therapeutic proteins into tissues and across the blood-brain barrier (BBB) is limited by the size and biochemical properties of the proteins. Efficient delivery across BBB is generally restricted to small, highly lipophilic molecules. However, in the last decades, several peptides that can pass cell membranes have been identified. It has been shown that these peptides are also capable of delivering large hydrophilic cargoes into cells and are therefore a powerful biological tool for transporting drugs across cell membranes and even into the brain. We designed and prepared a single-chain antibody fragment (scFvs), specific for the pathological form of the prion protein (PrP(Sc)), where a cell-penetrating peptide (CPP) was used as a linker between the two variable domains of the scFv. The intravenously administered recombinant scFv-CPP was successfully targeted to and delivered into mouse brain cells. Our single-chain antibody fragments are of special interest in view of possible therapeutic reagents design not only for prion diseases but also for other neurodegenerative diseases.

Specific binding of the pathogenic prion isoform: development and characterization of a humanized single-chain variable antibody fragment.

Murine monoclonal antibody V5B2 which specifically recognizes the pathogenic form of the prion protein represents a potentially valuable tool in diagnostics or therapy of prion diseases. As murine antibodies elicit immune response in human, only modified forms can be used for therapeutic applications. We humanized a single-chain V5B2 antibody using variable domain resurfacing approach guided by computer modelling. Design based on sequence alignments and computer modelling resulted in a humanized version bearing 13 mutations compared to initial murine scFv. The humanized scFv was expressed in a dedicated bacterial system and purified by metal-affinity chromatography. Unaltered binding affinity to the original antigen was demonstrated by ELISA and maintained binding specificity was proved by Western blotting and immunohistochemistry. Since monoclonal antibodies against prion protein can antagonize prion propagation, humanized scFv specific for the pathogenic form of the prion protein might become a potential therapeutic reagent.

Single-chain Fv antibody fragments retain binding properties of the monoclonal antibody raised against peptide P1 of the human prion protein.

Prion diseases are incurable neurodegenerative diseases that affect both humans and animals. The infectious agent is a pathogenic form of the prion protein that accumulates in brain as amyloids. Currently, there is neither cure nor reliable preclinical diagnostics on the market available. The growing number of reports shows that passive immunisation is one of the most promising strategies for prion disease therapy, where antibodies against prions may prevent and even cure the infection. Since antibodies are large molecules and, thus, might not be suitable for the therapy, different antibody fragments are a good alternative. Therefore, we have designed and prepared single-chain antibody fragments (scFvs) derived from the PrP(Sc)-specific murine monoclonal antibody V5B2. Using a new expression vector pMD204, we produced scFvs in two opposing chain orientations in the periplasm of Escherichia coli. Both recombinant antibody fragments retained the specificity of the parent antibody and one of these exhibited binding properties comparable to the corresponding murine Fab fragments with the affinity in nM range. Our monovalent antibody fragments are of special interest in view of possible therapeutic reagents for prion diseases as well as for development of a new generation of diagnostics.