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.     

Improvement of binding of Puumala virus neutralization site resembling peptide with a second-generation phage library

We have previously selected a peptide insert FPCDRLSGYWERGIPSPCVRrecognizing the Puumala virus (PUUV) G2-glycoprotein-specificneutralizing monoclonal antibody (MAb) 1C9 with K_d of 2.85x10^–8from a random peptide library X₂CX₁₄CX₂ expressed on the pIIIprotein of the filamentous phage fd-tet. We have now createda second-generation phage-displayed peptide library in whicheach amino acid of the peptide was mutated randomly to anotherwith a certain probability. Peptides were selected for higheraffinity for MAb 1C9 and for a common binding motif for MAb4G2 having an overlapping epitope with MAb 1C9 in G2 glycoprotein.The resulting peptides were synthesized as spots on cellulosemembrane. Amino acid changes which improved the reactivity ofthe peptides to MAb 1C9 were combined in the peptide ATCDKLFGYYERGIPLPCALwith K_d of 1.49x10^–9 in biosensor measurements. Our resultsshow that the binding properties of peptides, the affinity andthe specificity can be improved and the binding specificitydetermining amino acids and structural factors can be analyzedby combining binding assays with synthetic peptides on membranewith the use of second-generation phage display libraries. Received October 18, 2002; revised April 16, 2003; accepted May 26, 2003.     

Control of antibody-antigen interaction using anion-induced conformational change in antigen peptide

The binding of a monoclonal antibody to an epitope peptide wascontrolled by the conformational change of the epitope peptideinduced by anions. We synthesized peptides in which the epitopesequence DTYRYI for the monoclonal antibody AU1 is located betweenamphiphilic peptides (KKLL)_n and (LLKK)_n. In the absence ofan appropriate anion, the peptide was in a random coil stateand the epitope was linear. In contrast, in the presence ofan appropriate anion, the peptide exhibited an anti-parallel-helical structure and the epitope was subsequently `bent'.In the presence of 41 µM triphosphate, the associationconstant between the antibody and the peptide was decreasedby one order of magnitude in the case of n = 3 and at leastthree orders of magnitude in the case of n = 4 or 5. Oligo-DNAs,as anions, dissociated the antibody–peptide complex, whereastriphosphate did not. The DNA concentrations required for 50%dissociation of the antibody–peptide complex at pH 7.5were 4x10^–8, 1x10^–7 and 6x10^–6 M for decamer,octamer and hexamer DNA, respectively.     

Anti-c-myc antibody 9E10: epitope key positions and variability characterized using peptide spot synthesis on cellulose

The 9E10 antibody epitope (EQKLISEEDL) derives from a proteinsequence in the human proto-oncogen p62^c-myc and is widely usedas a protein fusion tag. This myc-tag is a powerful tool inprotein localization, immunochemistry, ELISA or protein purification.Here, we characterize the myc-tag epitope by substitutionalanalysis and length variation using peptide spot synthesis oncellulose. The key amino acids of this interaction are the coreresidues LISE. The shortest peptide with a strong binding signalis KLISEEDL. Dissociation constants of selected peptide variantsto the antibody 9E10 were determined. scFv constructs with theshortest possible myc-tags were successfully detected by Westernblot and ELISA, giving a signal comparable to that of the originalmyc-tag.     

Sulfolobus solfataricus protein disulphide oxidoreductase: insight into the roles of its redox sites

Sulfolobus solfataricus protein disulphide oxidoreductase (SsPDO)contains three disulphide bridges linking residues C⁴¹XXC⁴⁴,C¹⁵⁵XXC¹⁵⁸, C¹⁷³XXXXC¹⁷⁸. To get information on the role playedby these cross-links in determining the structural and functionalproperties of the protein, we performed site-directed mutagenesison Cys residues and investigated the changes in folding, stabilityand functional features of the mutants and analysed the resultswith computational analysis. The reductase activity of SsPDOand its mutants was evaluated by insulin and thioredoxin reductaseassays also coupled with peroxiredoxin Bcp1 of S. solfataricus.The three-dimensional model of SsPDO was constructed and correlatedwith circular dichroism data and functional results. Biochemicalanalysis indicated a key function for the redox site constitutedby Cys155 and Cys158. To discriminate between the role of thetwo cysteine residues, each cysteine was mutagenised and thebehaviour of the single mutants was investigated elucidatingthe basis of the electron-shuffling mechanism for SsPDO. Finally,cysteine pK values were calculated and the accessible surfacefor the cysteine side chains in the reduced form was measured,showing higher reactivity and solvent exposure for Cys155.     

Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45

By chemoenzymatic synthesis the gene for a (Leu²⁷) analogueof human growth hormone releasing hormone-Gly⁴⁵ [(Leu²⁷GHRH-Gly⁴⁵]was constructed, cloned and expressed in Escherichia coli asa fusion protein with ß-galactosidase under the controlof the lac promoter and operator. Upon induction with isopropyl-D-thio-ß-galactopyranosidethe fusion protein accumulated to a yield of 15–20% ofthe total cellular protein. After cyanogen bromide deavage ofthe fusion protein the precursor peptide (Leu²⁷)hGHRH-Gly⁴⁵was separated by extraction and purified by ion exchange andh.p.l.c.-RP18 chromatography. The purified peptide was analysedby sequencing, isolectric focusing, amino acid analysis andamino acid analysis after V8 protease digestion. The carboxy-terminalglydne was subsequently amidated by PAM (peptidylglycine--amidating-monooxygenase),an enzyme which was isolated and characterized from fresh bovinepituitaries. Correct amidatlon of the penultimate amino acid,leucine, was verified by peptide sequencing with an authenticleucine amide reference.     

Conversion of the guanine nucleotide binding sites of ras protein resulting in the reduction of base specificity

A gene coding for the novel ras protein, p21^x, in which thedomains of guanine binding and phosphate binding were exchanged,was constructed and expressed in Escherichia coli. The geneproduct, p21^x, showed GTP binding activity, but no GPTase activity.In addition, p21^x revealed binding activity toward ATP and CTP.In a competitive binding assay, [³H]GTP binding to p21^x wasinhibited in the presence of ATP, CTP and UTP, ITP as well asGDP, GTP and dGTP.     

QSAR studies applied to the prediction of antigen-antibody interaction kinetics as measured by BIACORE

The objective of this work was to investigate the potentialof the quantitative structure–activity relationships (QSAR)approach for predictive modulation of molecular interactionkinetics. A multivariate QSAR approach involving modificationsin peptide sequence and buffer composition was recently usedin an attempt to predict the kinetics of peptide–antibodyinteractions as measured by BIACORE. Quantitative buffer–kineticsrelationships (QBKR) and quantitative sequence–kineticsrelationships (QSKR) models were developed. Their predictivecapacity was investigated in this study by comparing predictedand observed kinetic dissociation parameters (k_d) for new antigenicpeptides, or in new buffers. The range of experimentally measuredk_d variations was small (300-fold), limiting the practical valueof the approach for this particular interaction. However, themodels were validated from a statistical point of view. In QSKR,the leave-one-out cross validation gave Q² = 0.71 for 24 peptides(all but one outlier), compared to 0.81 for 17 training peptides.A more precise model (Q² = 0.92) could be developed when removingsets of peptides sharing distinctive structural features, suggestingthat different peptides use slightly different binding modes.All models share the most important factor and are informativefor structure–kinetics relationships. In QBKR, the measuredeffect on k_d of individual additives in the buffers was consistentwith the effect predicted from multivariate buffers. Our resultsopen new perspectives for the predictive optimization of interactionkinetics, with important implications in pharmacology and biotechnology.     

A dual-affinity gene fusion system to express small recombinant proteins in a soluble form: expression and characterization of protein A deletion mutants

A novel gene fusion system to express and purify small recombinantproteins in Escherichia coli has been constructed. The conceptallows for affinity purification of soluble gene products bysequential albumin- and Zn²+-affinity chromatography. The dual-affinitysystem is well suited for expression of unstable proteins asonly full-length protein is obtained after purification andproteins gain proteolytic stability in the fusion protein. Herewe show that the dual-affinity approach can be used for theexpression of various unstable derivatives of a single IgG-bindingdomain based on staphylococcal protein A. Analysis of the proteolyticstabilities and the IgG-binding properties of the differentmutant proteins suggest that the model for the structure ofan IgG-binding domain must be re-evaluated.     

Progesterone binding to uteroglobin: two alternative orientations of the ligand

Progesterone binding to a homodimer of uteroglobin takes placein a hydrophobic cavity formed by the two subunits. Previousmutational analyses have shown that the tyrosine (21 and 21')and threonine (60 and 60') residues of the uteroglobin dimerare directly involved in progesterone binding. To analyze thecontribution of each of the two tyrosines and threonines inthe dimer, we have constructed a covalently linked uteroglobindimer (UG^cl) by fusing two uteroglobin cDNAs via a syntheticlinker sequence. Escherichia coli expressed UG^cl bound progesteronewith the same affinity as the native dimeric protein. Replacementof both tyrosines by phenylalanines abolished progesterone binding.Replacement of either the C-terminal tyrosine 21 or the N-terminaltyrosine 21' separately, reduced the affinity for progesterone3- to 4-fold, suggesting that both tyrosines participate inprogesterone binding. In contrast, substitutions of the threonineresidues of the C- or Nterminal moities had no effect, whereasthe replacement of both threonines reduced the affinity forprogesterone 2- to 3-fold. These data, together with computermodels, suggest that progesterone docks in the internal bindingpocket of uteroglobin in two different orientations.     

In vitro scanning saturation mutagenesis of all the specificity determining residues in an antibody binding site

For the first time, each specificity determining residue (SDR)in the binding site of an antibody has been replaced with everyother possible single amino acid substitution, and the resultingmutants analyzed for binding affinity and specificity. The studieswere conducted on a variant of the 26-10 antidigoxin singlechain Fv (scFv) using in vitro scanning saturation mutagenesis,a new process that allows the high throughput production andcharacterization of antibody mutants [Burks,E.A., Chen,G., Georgiou,G.and Iverson,B.L. (1997) Proc. Natl Acad. Sci. USA, 94, 412–417].Single amino acid mutants of 26-10 scFv were identified thatmodulated specificity in dramatic fashion. The overall plasticityof the antibody binding site with respect to amino acid replacementwas also evaluated, revealing that 86% of all mutants retainedmeasurable binding activity. Finally, by analyzing the physicalproperties of amino acid substitutions with respect to theireffect on hapten binding, conclusions were drawn regarding thefunctional role played by the wild-type residue at each SDRposition. The reported results highlight the value of in vitroscanning saturation mutagenesis for engineering antibody bindingspecificity, for evaluating the plasticity of proteins, andfor comprehensive structure–function studies and analysis. ⁴ To whom correspondence should be addressed     

Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling

The possibility of increasing the affinity of a Taq DNA polymerasespecific binding protein (affibody) was investigated by an -helixshuffling strategy. The primary affibody was from a naive combinatoriallibrary of the three-helix bundle Z domain derived from staphylococcalprotein A. A hierarchical library was constructed through selectivere-randomization of six amino acid positions in one of the two-helices of the domain, making up the Taq DNA polymerase bindingsurface. After selections using monovalent phage display technology,second generation variants were identified having affinities(K_D) for Taq DNA polymerase in the range of 30–50 nM asdetermined by biosensor technology. Analysis of binding dataindicated that the increases in affinity were predominantlydue to decreased dissociation rate kinetics. Interestingly,the affinities observed for the second generation Taq DNA polymerasespecific affibodies are of similar strength as the affinitybetween the original protein A domain and the Fc domain of humanimmunoglobulin G. Further, the possibilities of increasing theapparent affinity through multimerization of affibodies wasdemonstrated for a dimeric version of one of the second generationaffibodies, constructed by head-to-tail gene fusion. As comparedwith its monomeric counterpart, the binding to sensor chip immobilizedTaq DNA polymerase was characterized by a threefold higher apparentaffinity, due to slower off-rate kinetics. The results showthat the binding specificity of the protein A domain can bere-directed to an entirely different target, without loss ofbinding strength.     

Site-directed mutants of the cAMP receptor protein-DNA binding of five mutant proteins

Oligonucleotide-directed mutagenesis was employed to generatemutants of the cAMP receptor protein (CRP) of Escherichia coli.The mutant proteins were purified to homogeneity and testedfor stability and DNA binding. It is shown that mutations atthe position of Arg¹⁸⁰ abolish specific DNA binding, whereasthose at the position Arg¹⁸⁵ have very little effect. Both positionshave previously been implicated as crucial for the specificinteraction between CRP and DNA. The Ser¹²⁸ Ala mutant showsa slight reduction in DNA binding affinity relative to wild-type.All mutants investigated show similar stability profiles towild-type CRP with respect to thermolysin proteolysis as a functionof temperature.     

Expression of mouse immunoglobulin light and heavy chain variable regions in Escherichia coli and reconstitution of antigen-binding activity

The expression of immunoglobulin heavy and light chain variableregions in the cytoplasm of Escherichia coli and formation ofa functional heterodimer has been demonstrated. Variable domainsequences were taken from the heavy and light chain cDNAs ofthe monoclonal antibody Gloop 2 and engineered for expressionin a dual origin expression vector. The engineered genes vhg2and vlg2 were separately subcloned into the vector, creatingtwo expression plasmids. Expression of the heavy and light chainvariable region genes (encoding 116 and 109 amino adds respectively)was investigated in eight E.coli strains; the polypeptides wererapidly degraded in a host strain optimized for expression andin E.coli strains deficient in the major protease La (lon^-).Accumulation was permitted in severely protease-deficient E.colihaving a defective heat-shock response. A lon^- mutation in thisgenetic background permitted even higher accumulation. Expressionlevels were 7 and 1% of total bacterial protein for light andheavy chain variable regions respectively. Expression of theheavy chain variable region gene was increased by includinga longer Shine–Dalgarno sequence. Similar constructionsin the light chain vector had no effect on expression levels.The insoluble variable region polypeptides were reconstitutedinto a heterodimer possessing the full antigen binding characteristicsof both the parent monoclonal antibody and its Fab fragment.     

3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity

The substitution of aspartate at position 153 in Escherichiacoli alkaline phosphatase by glycine results in a mutant enzymewith 5-fold higher catalytic activity (k_cat but no change inKm at pH 8.0 in 50 mM Tris-HCl. The increased k_cat is achievedby a faster release of the phosphate product as a result ofthe lower phosphate affinity. The mutation also affects Mg²⁺binding, resulting in an enzyme with lower metal affinity. The3-D X-ray structure of the D153G mutant has been refined at2.5 Å to a crystallographic Rfactor of 16.2%. An analysisof this structure has revealed that the decreased phosphateaffinity is caused by an apparent increase in flexibility ofthe guanidinium side chain of Argl66 involved in phosphate binding.The mutation of Aspl53 to Gly also affects the position of thewater ligands of Mg²⁺, and the loop Glnl52–Thrl55 is shiftedby 0.3 Å away from the active site. The weaker Mg²⁺ bindingof the mutant compared with the wild type is caused by an alteredcoordination sphere in the proximity of the Mg²⁺ ion, and alsoby the loss of an electrostatic interaction (Mg²⁺.COO^-Aspl53)in the mutant Its ligands W454 and W455 and hydroxyl of Thrl55,involved in the octahedral coordination of the Mg²⁺ ion, arefurther apart in the mutant compared with the wild-type     

Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus

HNK20 is a mouse monoclonal IgA that binds to the F glycoproteinof respiratory syncytial virus (RSV) and neutralizes the virus,both in vitro and in vivo. The single-chain antibody fragment(scFv) derived from HNK20 is equally active and has allowedus to assess rapidly the effect of mutations on affinity andantiviral activity. Humanization by variable domain resurfacingrequires that surface residues not normally found in a humanFv be mutated to the expected human amino acid, thereby eliminatingpotentially immunogenic sites. We describe the constructionand characterization of two humanized scFvs, hu7 and hu10, bearing7 and 10 mutations, respectively. Both molecules show unalteredbinding affinities to the RSV antigen (purified F protein) asdetermined by ELISA and surface plasmon resonance measurementsof binding kinetics (K_a 1x10⁹ M^–1). A competition ELISAusing captured whole virus confirmed that the binding affinitiesof the parental scFv and also of hu7 and hu10 scFvs were identical.However, when compared with the original scFv, hu10 scFv wasshown to have significantly decreased antiviral activity bothin vitro and in a mouse model. Our observations suggest thatbinding of the scFv to the viral antigen is not sufficient forneutralization. We speculate that neutralization may involvethe inhibition or induction of conformational changes in thebound antigen, thereby interfering with the F protein-mediatedfusion of virus and cell membranes in the initial steps of infection.     

Interactions of (Ala*Ala*Lys*Pro)n and (Lys*Lys*Ser*Pro)n with DNA. Proposed coiled-coil structure of AlgR3 and AlgP from Pseudomonas aeruginosa

The proteins, AlgR3 and AlgP, are involved in the regulationof alginate synthesis in Pseudomonas. They contain multiplerepeats of Ala^*Ala^*Lys^*Pro as do several other proteins thatresemble histones. The interactions of synthesis oligopeptidescomposed of repeated Ala^*Ala^*Lys^*Pro or Lys^*Lys^*Ser^*Pro unitswith DNA were studied by fluorescence of the Fmoc (9-fluorenylmethyloxycarbonyl)group attached to the N-termini of the peptides. DNA quenchingof the Fmoc fluorescence of the peptides was used to estimatethe apparent association constants for the interaction of Fmoc(AAKP)_nOH(n = 2, 4, 8, 18, 32) and of Fmoc(KKSP)_nOH (n = 2, 4, 8, 16,20, 32) with DNA. The Fmoc(AAKP)_nOH peptides bind to DNA onlyat low ionic strength; the Fmoc(KKSP)_n OH peptides interactwith DNA at both low (0.05 M KCl) and high (0.2 M KCl) saltAt low ionic strength an increase in the number of the repeatunits causes an increase in the apparent association constantup to {small tilde}2 x 10⁶ M^–1 for both types of peptidesat N 24. The insertion of an AAKTA unit into the middle ofthe Fmoc(AAKP)₈OH peptide increases its affinity to DNA. Wepropose a model of (AAKP)_n and of its interaction with DNA.The repeat unit consists of a single turn of -helix followedby a bend necessitated by Pro. The resultant coiled-coil formsa right-handed superhelix with 10 AAKPs per repeat distanceof {small tilde}33 Å. With only slight modification ofthe canonical parameters of this model the AAKP super helixfits into the major groove of B-form DNA with one AAKP tetramerper base pair repeat of 3.4 Å. The -amine nitrogen ofLys can form a polar hydrogen bond with a phosphate oxygen atomof the DNA backbone. A better fit is obtained when the modelis modified to accommodate [(AAKP)₅AAKTA]_n as actually observedin AlgR3. We suggest that this coiled-coil represents a generalmotif for other protein–DNA interactions.     

Structure-oriented rational design of chymotrypsin inhibitor models

Three peptides modelling a highly potent, 35-residue chymotrypsininhibitor (Schistocerca gregaria chymotrypsin inhibitor) weredesigned and synthesized by convergent peptide synthesis. Foreach model peptide, the inhibitory constant (K_i) on chymotrypsinand the solution structure were determined. In addition, moleculardynamics calculations were performed for all of them. Two modelscontaining approximately half of the parent inhibitor (17 of35 residues) were designed and subsequently found to have nosubstantial inhibitory activity (K_i values in the mM range).The third model composed of 24 amino acid residues proved tobe an effective (K_i 10^–7) inhibitor of bovine chymotrypsin.Both the solution structure properties determined by NMR spectroscopyand the dynamic behaviour of the latter model system are comparableto the native inhibitor. In contrast, the structure and dynamicsof the first two related model peptides show characteristicdifferences. We suggest that the conformation and flexibilityof the modelled protease inhibitor are crucial for its biologicalefficiency. Moreover, the structural and dynamic features ofthe binding loop (28–33) and those of the rest of themolecule appear to be interdependent. Most importantly, thesestructural characteristics can be rationally modified, at leastpartially, by peptide design. Received March 7, 2003; revised August 25, 2003; accepted August 26, 2003.