Properties of a single-chain antibody containing different linker peptides

Single-chain antibodies were constructed using six differentlinker peptides to join the V_H and V_L domains of an anti-2-phenyloxazolone(Ox) antibody. Four of the linker peptides originated from theinterdomain linker region of the fungal cellulase CBHI and consistedof 28, 11, six and two amino acid residues. The two other linkerpeptides used were the (GGGGS)₃ linker with 15 amino acid residuesand a modified IgG2b hinge peptide with 22 residues. Proteolyticstability and Ox binding properties of the six different scFvderivatives produced in Escherichia coli were investigated andcompared with those of the corresponding Fv fragment containingno joining peptide between the V domains. The hapten bindingproperties of different antibody fragments were studied by ELISAand BIAcore^TM. The interdomain linker peptide improved the haptenbinding properties of the antibody fragment when compared withFv fragment, but slightly increased its susceptibility to proteases.Single-chain antibodies with short CBHI linkers of 11, six andtwo residues had a tendency to form multimers which led to ahigher apparent affinity. The fragments with linkers longerthan 11 residues remained monomeric.     

Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment

Europium chelates provide a non-radioactive alternative forsensitive labelling of antibodies for diagnostic immunoassays.Lysine residues at antibody surfaces are ready targets for labellingby an isothiocyanate derivative of the europium chelate (Eu³⁺).Here the labelling efficiency of a recombinant anti-human -fetoprotein(hAFP) Fab fragment has been improved by increasing its lysinecontent by protein engineering. Molecular modelling was usedto identify three light chain constant domain surface arginineresidues, R154, R187 and R210, which were mutated to lysineresidues. The mutations did not influence the affinity of thelysine-enriched Fab fragment and its labelling efficiency wasfound to be 40% higher than that of the wildtype Fab fragmentWith low degree of labelling, the affinities of the two Fabfragments were identical and comparable with that of the originalmonoclonal anti-hAFP IgG. With a higher degree of labellingthe affinities of both Fab fragments decreased more than thatof the intact IgG since more lysine residues are available forlabelling in the additional heavy chain constant domains ofthe larger molecule. Electrostatic adsorption and covalent immobilizationof the Fab fragments were characterized by BIAcore^TM and thelysine-enriched Fab fragment was found to be more efficientlyimmobilized to an activated carboxymethyl surface.     

An active single-chain antibody containing a cellulase linker domain is secreted by Escherichia coli

Single-chain antibodies consist of the variable, antigen-bindingdomains of antibodies joined to a continuous polypeptide bygenetically engineered peptide linkers. We have used the flexibleinterdomain linker region of a fungal cellulase to link togetherthe variable domains of an anti-2-phenyloxazolone IgGl and showhere that the resulting single-chain antibody is efficientlysecreted and released to the culture medium of Escherichia coli.The yield of affinity-purified single-chain antibody is 1 -2mg/1 of culture medium and its affinity and stability are comparableto those of the corresponding native IgG.     

Abrasive wear of steel against gravel with different rock–steel combinations

Wear testing equipment and tests used in research laboratories are often miniature or simplified versions of real applications. For example standardized ASTM dry sand rubber wheel abrasion test G 65 and pin abrasion test G 132 are widely used to study materials’ abrasion wear resistance. The test results, however, do not always correlate too well with the results obtained from real wear conditions. One reason for this is, for example, that in the crushing applications of mining industry the abrasive size is usually much larger than that used in the laboratory wear tests. To study the abrasive wear caused by larger size gravel, new three-body abrasion test equipment was therefore constructed. The equipment uses the pin-on-disk principle with free abrasive particles of sizes up to 10 mm. During the test the pin is repeatedly pressed against a fixed amount of abrasive that is rotating with the disk having confining walls. As the pin is prevented from touching the counterbody, only the abrasive acts as the wearing agent.Three steels of different hardnesses were cross-tested as pin–disk pairs and as pins against a rubber disk using three igneous rock gravels with different crushability properties as abrasives. The wear was measured as mass loss from both the pin and the disk, and the rock comminution was measured by sieving. The results indicate that the mechanism of wear is greatly affected by the hardness of the counterbody. When using large size abrasives, the rate of comminution is also a very important factor that can significantly affect the wear test results.     

A scheme for designating enzymes that hydrolyse the polysaccharides in the cell walls of plants

Twelve neurologically normal infants (age 2.9+/-0.9 months) with peptic esophagitis (grade 2) who did not respond to cimetidine (in addition to positioning, cisapride, and Gaviscon) were treated with omeprazole, 0.5 mg/kg once a day, for 6 weeks. The effectiveness of omeprazole was evaluated in all infants by clinical assessment and endoscopy before and after treatment and by 24-hour gastric pH monitoring during treatment in seven infants. Omeprazole therapy led to a marked decrease in symptoms, endoscopic and histologic signs of esophagitis, and intragastric acidity.     

Molecular dynamics simulation of fungal cellulose-binding domains: differences in molecular rigidity but a preserved cellulose binding surface

A total of 23 fungal cellulose-binding domain (CBD) sequenceswere aligned. Structural models of the cellulosebinding domainof an exoglucanase (CBHII) and of three endoglucanases (EGI,EGII and EGV) from Trichoderma reesei cellulases were homologymodelled based on the NMR structure of the fungal cellobiohydrolaseCBHI, from the same organism. The completed models and the knownstructure of the CBHI cellulose-binding domain were refinedby molecular dynamics simulations in water. All four modelswere found to be very similar to the structure of the CBHI cellulose-bindingdomain and sequence comparison indicated that in general thethree-dimensional structures of fungal cellulose-binding domainsare very similar. In all the CBDs studied, two disulphide bridgesapparently stabilize the polypeptide fold. From the models,an additional disulphide bridge was predicted in EGI and CBHII,and in eight further CBDs from other organisms. Three highlyconserved aromatic residues on the hydrophilic side of the wedgemake this surface flat This surface is expected to make contactwith the substrate. Three invariant amino acids, Gln7, Asn29and Gln34, on this flat face are in suitable positions for hydrogenbonding with the cellulose surface. Analysis of the differencesin the protein surface properties indicated that the endoglucanasestend to be more hydrophilic than the exoglucanases. The largeststructural variation was found around positions 12-16. The fungalCBD sequences are discussed in relation to variations in functionand pH dependence. Comparison of the modelled structures withexperimental binding data for the CBHI and EGI allowed the formulationof a qualitative relationship to cellulose affinity. This relationshipwas used to predict the cellulose affinities for 21 CBDs.     

The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169

Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanasecleaving primarily cellobiose units from the non-reducing endof cellulose chains. The ß-l,4 glycosidic bond iscleaved by acid catalysis with an aspartic acid, D221, as thelikely proton donor, and another aspartate, D175, probably ensuringits protonation and stabilizing charged reaction intermediates.The catalytic base has not yet been identified experimentally.The refined crystal structure of CBHII also shows a tyrosineresidue, Y169, located close enough to the scissile bond tobe involved in catalysis. The role of this residue has beenstudied by introducing a mutation Y169F, and analysing the kineticand binding behaviour of the mutated CBHII. The crystal structureof the mutated enzyme was determined to 2.0 Å resolutionshowing no changes when compared with the structure of nativeCBHII. However, the association constants of the mutant enzymefor cellobiose and cellotriose are increased threefold and for4-methylumbelliferyl cellobioside over 50-fold. The catalyticconstants towards cellotriose and cellotetraose are four timeslower for the mutant. These data suggest that Y169, on interactingwith a glucose ring entering the second subsite in a narrowtunnel, helps to distort the glucose ring into a more reactiveconformation. In addition, a change in the pH activity profilewas observed. This indicates that Y169 may have asecond rolein the catalysis, namely to affect the protonation state ofthe active site carboxylates, D175 and D221.     

Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris

Fusion proteins composed of a cellulose-binding domain fromNeocallimastix patriciarum cellulase A and Candida antarcticalipase B were constructed using different linker peptides. Theaim was to create proteolytically stable linkers that were ableto join the functional modules without disrupting their function.Six fusion variants containing linkers of 4–44 residueswere expressed in Pichia pastoris and analysed. Three variantswere found to be stable throughout 7-day cultivations. The cellulose-bindingcapacities of fusion proteins containing short linkers wereslightly lower compared with those containing long linkers.The lipase-specific activities of all variants, in solutionor immobilized on to cellulose, were equal to that of the wild-typelipase.     

High-resolution crystal structures reveal how a cellulose chain is bound in the 50 A long tunnel of cellobiohydrolase I from Trichoderma reesei

Detailed information has been obtained, by means of protein X-ray crystallography, on how a cellulose chain is bound in the cellulose-binding tunnel of cellobiohydrolase I (CBHI), the major cellulase in the hydrolysis of native, crystalline cellulose by the fungus Trichoderma reesei. Three high-resolution crystal structures of different catalytically deficient mutants of CBHI in complex with cellotetraose, cellopentaose and cellohexaose have been refined at 1.9, 1.7 and 1.9 A resolution, respectively. The observed binding of cellooligomers in the tunnel allowed unambiguous identification of ten well-defined subsites for glucosyl units that span a length of approximately 50 A. All bound oligomers have the same directionality and orientation, and the positions of the glucosyl units in each binding site agree remarkably well between the different complexes. The binding mode observed here corresponds to that expected during productive binding of a cellulose chain. The structures support the hypothesis that hydrolysis by CBHI proceeds from the reducing towards the non-reducing end of a cellulose chain, and they provide a structural explanation for the observed distribution of initial hydrolysis products.