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Hemminki Ari; Hoffren Anna-Marja; Takkinen Kristiina; Vehniainen Markus; Makinen Maija-Liisa; Pettersson Kim; Teleman Olle; Soderlund Hans; Teeri Tuula T. 《Protein engineering, design & selection : PEDS》1995,8(2):185-191
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 BIAcoreTM and thelysine-enriched Fab fragment was found to be more efficientlyimmobilized to an activated carboxymethyl surface. 相似文献
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Hoffren Anna-Marja; Saloheimo Markku; Thomas Pamela; Overington John P.; Johnson Mark S.; Knowles Jonathan K.C.; Blundell Tom L. 《Protein engineering, design & selection : PEDS》1993,6(2):177-182
A model of the lignin peroxidase LIII of Phlebia radiata wasconstructed on the basis of the structure of cytochrome c peroxidase(CCP). Because of the low percentage of amino acid identitybetween the CCP and the lignin peroxidase LIII of Phlebia radiata,alignment of the sequences was based on the generation of atemplate from a knowledge of the 3-D structure of CCP and consensussequences of lignin peroxidases. This approach gave an alignmentin which all the insertions in the lignin peroxidase were placedat loop regions of CCP, with a 21.1% identity for these twoproteins. The model was constructed using this alignment andthe computer program COMPOSER, which assembles the model asa series of rigid fragments derived from CCP and other proteins.Manual intervention was required for some of the longer loopregions. The -helices forming the structural framework, andespecially the haem environment of CCP, are conserved in theLIII model and the core is close packed without holes. A possiblesite of the substrate oxidation at the haem edge of LIII isdiscussed. 相似文献
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Molecular dynamics simulation of fungal cellulose-binding domains: differences in molecular rigidity but a preserved cellulose binding surface 总被引:1,自引:0,他引:1
Hoffren Anna-Marja; Teeri Tuula T.; Teleman Olle 《Protein engineering, design & selection : PEDS》1995,8(5):443-450
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. 相似文献
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Specificity improvement of a recombinant anti-testosterone Fab fragment by CDRIII mutagenesis and phage display selection 总被引:1,自引:0,他引:1
Hemminki A; Niemi S; Hoffren AM; Hakalahti L; Soderlund H; Takkinen K 《Protein engineering, design & selection : PEDS》1998,11(4):311-319
The monoclonal antibodies so far developed by hybridoma technology have not
had high enough specificity or affinity to distinguish the closely related
steroid hormones in routine clinical assays. We have employed random
mutagenesis and phage display approaches to improve the specificity of one
anti-testosterone monoclonal antibody (3-C4F5). The affinity of the
antibody is 0.3 x 10(9) M(-1) and the cross- reactivities with most of the
related steroids are low. However, the antibody cross-reacts about 1% with
dehydroepiandrosterone sulfate (DHEAS) and owing to the high DHEAS serum
concentration this is about 1000-fold too high for clinical immunoassays.
The complementarity- determining regions (CDRs) of the heavy and light
chains, which were predicted by molecular modelling to be in close contact
with the testosterone (TES) ligand, were randomized and mutant Fab
libraries were cloned into a phagemid vector. Binders were selected by a
competitive panning procedure. By combining the identified light and heavy
chain CDRIII mutations the TES affinity was preserved at the wild- type
level but DHEAS cross-reactivity was decreased to 0.03%. An important
finding was that by the competitive panning procedure the overall binding
specificity of the 3-C4F5 antibody was refined, since the
cross-reactivities to related steroids were also significantly decreased in
the combined mutant.
相似文献
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