Targeting myeloid leukemia with a DT390-mIL-3 fusion immunotoxin: ex vivo and in vivo studies in mice

The IL-3 receptor was expressed on a high frequency of myeloidleukemia cells and also on hematopoietic and vascular cells.We previously showed that a recombinant IL-3 fusion immunotoxin(DT₃₉₀IL-3) expressed by splicing the murine IL-3 gene to atruncated diphtheria toxin (DT₃₉₀) gene selectively killed IL-3R⁺expressing cells and was not uniformly toxic to uncommited BMprogenitor cells (Chan,C.-H., Blazar,B.R., Greenfield,L., Kreitman,R.J.and Vallera,D.A., 1996, Blood, 88, 1445–1456). Thus, weexplored the feasability of using DT₃₉₀IL-3 as an anti-leukemiaagent. DT₃₉₀IL-3 was toxic when administered to mice at dosesas low as 0.1 µg/day. The dose limiting toxicity appearedto be related to platelet and bleeding effects of the fusiontoxin. Because of these effects, DT₃₉₀IL-3 was studied ex vivoas a means of purging contaminating leukemia cells from BM graftsin a murine autologous BM transplantation. In this setting,as few as 1000 IL-3R-expressing, bcr/abl transformed myeloid32Dp210 leukemia cells were lethal. An optimal purging intervalof 10 nM/l for 8 h eliminated leukemia cells from 32Dp210/BMmixtures given to lethally irradiated (8 Gy) C3H/HeJ syngeneicmice. Mice given treated grafts containing BM and a lethal doseof 32Dp210 cells survived over 100 days while mice given untreatedgrafts did not survive (P < 0.00001). DT₃₉₀IL-3 may provehighly useful for ex vivo purging of lethal malignant leukemiacells from autologous BM grafts.     

Bacterial expression, purification and preliminary kinetic description of the kinase domain of v-fps

The gene coding for the tyrosine protein kinase domain of v-fpswas subcloned into a plasmid vector expressing glutathione-S-transferase(GST). This new vector expresses a fusion protein in Escherichiacoli composed of the kinase domain linked with GST at the N-terminus(GST-kin). A portion of the total expressed protein was solubleupon cell lysis and was purified by affinity chromatographyusing glutathione cross-linked agarose. GST-kin (M_r 57 000)is a phosphoprotein as judged by ³²P autoradiography, consistentwith the known autophosphorylation site within the kinase core[Weinmaster et aL (1984) Cell, 37, 559–568]. Cleavageof the fusion protein with thrombin and purification on phosphocelluloseresin yielded the pure kinase domain (M_r 33 000). The activityof the kinase domain is indistinguishable from that of GST-kinusing the peptide substrate EEEIYEEIE, indicating that Nterminalfusion has no effect on the kinase domain. GSTkin phosphorylatesa second peptide, EAEIYEAIE, with improved catalytic efficiency.Initial velocity data are consistent with a random bireactantmechanism with no substrate synergism observed in the ternarycomplex. Steady-state kinetic analyses reveal that this peptideis phosphorylated, with a k_cat of 3.6 s^–1, a K_peptideof 500 µM and a K_ATP of 250 µM. The expression,purification and preliminary kinetic analysis of the kinasedomain of v-fps provide the first step in the application ofstructurefunction studies for this oncoprotein     

Selection of a thermostable variant of chloramphenicol acetyltransferase (Cat-86)

The moderate thermophile Bacillus stearothermophilus was usedas a host in which to detect more thermostable variants of theB.pumilus chloramphenicol acetyltransferase (Cat-86) protein.Seventeen mutants were isolated and detected by their abilityto grow in the presence of chloramphenicol at a previously restrictivetemperature (58°C). The genes encoding these proteins weresequenced; all 17 mutants carried the same C to T transitionthat conferred an amino acid substitution of alanine by valineat position 203 of the protein sequence. The wild-type and onemutant Cat-86 protein were purified to homogeneity using affinitychromatography, and kinetic and thermal stability studies wereundertaken. Both enzymes had similar sp. act. in the regionof 215 U/mg, with K_m values for chloramphenicol in the range13.8–15.4 µM and for acetyl CoA in the range 13.6–15.5µM. The A203V mutant shows greater stability than thewild-type Cat-86 protein at temperatures above 50°C andappears to pass through a transition state between 48 and 50°C.     

Chemical engineering of a three-fingered toxin with anti-{alpha}7 neuronal acetylcholine receptor activity

Though it possesses four disulfide bonds the three-fingeredfold is amenable to chemical synthesis, using a Fmoc-based method.Thus, we synthesized a three-fingered curaremimetic toxin fromsnake with high yield and showed that the synthetic and nativetoxins have the same structural and biological properties. Bothwere characterized by the same 2D NMR spectra, identical highbinding affinity (K_d = 22 ± 5 pM) for the muscular acetylcholinereceptor (AChR) and identical low affinity (K_d = 2.0 ±0.4 µM) for     

Fluorolabeling of antibody variable domains with green fluorescent protein variants: application to an energy transfer-based homogeneous immunoassay

A site-specific and efficient fluorolabeling of antibody variableregions with green fluorescent protein (GFP) variants and itsapplication to an energy transfer-based homogeneous fluoroimmunoassay(open sandwich FIA) were attempted. Two chimeric proteins, Trx–V_H–EBFPand Trx–V_L–EGFP, consisting of V_H and V_L fragmentsof anti-hen egg lysozyme (HEL) antibody HyHEL-10 and two GFPcolor variants, EBFP and EGFP, respectively, were designed tobe expressed in cytoplasm of trxB – mutant Escherichiacoli as fusions with thioredoxin from E.coli The mixture oftwo proteins could be purified with HEL-affinity chromatography,retaining sufficient intrinsic fluorescence and binding activityto HEL. A significant increase in fluorescence resonance energytransfer (FRET) dependent on HEL concentration was observed,indicating the reassociation of the V_H and V_L domains of thesechimeric proteins due to co-existing antigen. With this opensandwich FIA, an HEL concentration of 1–100 µg/mlcould be non-competitively determined. The assay could be performedin a microplate format and took only a few minutes to obtaina sufficient signal after simple mixing of the chimeric proteinswith samples. This represents the first demonstration that theFRET between GFP variants is applicable to homogeneous immunoassay.     

On the possibilities and limitations of rational protein design to expand the specificity of restriction enzymes: a case study employing EcoRV as the target

The restriction endonuclease EcoRV has been characterized instructural and functional terms in great detail. Based on thisdetailed information we employed a structure-guided approachto engineer variants of EcoRV that should be able to discriminatebetween differently flanked EcoRV recognition sites. In crystalstructures of EcoRV complexed with d(CGGGATATCCC)₂ and d(AAAGATATCTT)₂,Lys104 and Ala181 closely approach the two base pairs flankingthe GATATC recognition site and thus were proposed to be a reasonablestarting point for the rational extension of site specificityin EcoRV [Horton,N.C. and Perona,J.J. (1998) J. Biol. Chem.,273, 21721–21729]. To test this proposal, several single(K104R, A181E, A181K) and double mutants of EcoRV (K104R/A181E,K104R/A181K) were generated. A detailed characterization ofall variants examined shows that only the substitution of Ala181by Glu leads to a considerably altered selectivity with botholigodeoxynucleotide and macromolecular DNA substrates, butnot the predicted one, as these variants prefer cleavage ofa TA flanked site over all other sites, under all conditionstested. The substitution of Lys104 by Arg, in contrast, whichappeared to be very promising on the basis of the crystallographicanalysis, does not lead to variants which differ very much fromthe EcoRV wild-type enzyme with respect to the flanking sequencepreferences. The K104R/A181E and K104R/A181K double mutantsshow nearly the same preferences as the A181E and A181K singlemutants. We conclude that even for the very well characterizedrestriction enzyme EcoRV, properties that determine specificityand selectivity are difficult to model on the basis of the availablestructural information.     

Strong inhibition of fibrinogen binding to platelet receptor {alpha}IIb{beta}3 by RGD sequences installed into a presentation scaffold

In order to probe the structural constraints on binding of RGDsequences to the platelet receptor _IIbß₃ we have usedrecombinant DNA techniques to install the RGD sequence into‘presentation scaffolds’, small proteins of known3-D structure chosen to present guest sequences in constrainedorientations. Using Escherichia coli expression systems we madesequence variants in which loop residues of the immunoglobulinV_L domain REI and of human interleukin-1ß were replaced(without changing polypeptide length) by the RGD sequence atpositions predicted, based on small molecule studies, to orientthe RGD moiety into an active conformation. These variants donot compete for fibrinogen binding to _IIbß₃ up toalmost 1 mM concentration. Unfolded or proteolytically fragmentedforms of these same proteins do compete, however, showing thatthe RGD sequences in the mutants must be prohibited from bindingby constraints imposed by scaffold structure. To suppress theeffects of such structural constraints we constructed two sequencevariants in which RGD-containing sequences 42–57 or 44–55from the snake venom platelet antagonist kistrin were inserted(this increasing the length of the loop) into the third complementaritydetermining loop of REI. Both of these variants compete stronglyfor fibrinogen binding with IC₅₀s in the nM range. These results,plus data on kistrin-related peptides also presented here, suggestthat the molecular scaffold REI is capable of providing to aninstalled sequence a structural context and conformation beneficialto binding. The results also suggest that in order to bind wellto _IIbß₃, RGD sequences in protein ligands must eitherproject significantly from the surface of the scaffold and/orretain a degree of conformational flexibility within the scaffold.Molecular scaffolds like REI should prove useful in the elucidationof structure-function relationships and the discovery of newactive sequences, and may also serve as the basis for noveltherapeutic agents.     

Protien side-chain conformational entropy derived from fusion data-comparison with other empirical scales

The loss of conformational entropy of protein side-chains isa major effect in the energetics of folding. The simplest approachis to enumerate the number of freely rotatable bonds. Recently,two scales of side-chain conformational entropy have been proposedbased on the definition of entropy as the Boltzmann samplingover all accessible states (S –Rp_iInp_i, where p_i is theprobability of being in a rotameric state). In one scale, derivedonly for aliphatic and aromatic side-chains, the values of p_iwere obtained from Monte Carlo simulations. In the other scale,the observed frequencies of different rotameric states in adatabase of protein crystal structures yielded an estimate forp_i. Here an empirical estimation of the fusion entropy of theside-chains is used to derive a third scale. The fusion entropyis obtained as a sum of empirically derived contributions fromcomponent hydrocarbon and functional groups. There is a goodagreement between the fusion scale and the other two scales.This suggests that the magnitude of conformational entropy isbeing correctly established     

Expression of catalytically active hamster dihydroorotase domain in Escherichia coli: purification and characterization

Dihydroorotase is the central domain of trifunctional L-dihydroorotatesynthetase which also contains carbamyl phosphate synthetaseat the N-terminus and aspartate transcarbamylase at the C-terminus.The cDNA, corresponding to the active dihydroorotase domainas isolated after digestion of dihydroorotate synthetase withelastase, has been sub-cloned into the expression vector pCW12which was then used to transform Escherichia coli SØ1263pyrC^– lacking dihydroorotase activity. However, inductionof this recomhinant strain with IPTG produced large amountsof the dihydroorotase domain which were completely inactive.A number of cDNAs were expressed which were longer on the C-terminalside; all cDNAs expressed active dihydroorotase domain downto a minimal extension of 12 ammo adds (-Val- Pro-Pro-Gly-Tyr-GIy-Gm-Asp-Val-Arg-Lys-Trp)into the bridge region between the dihydroorotase and aspartatetranscarbamylase domains. Part of this dodecapeptide may forman amphipathk helix which in some way constrains the isolated,recombinant dihydroorotase domain to an active conformation.The recombinant hamster dihydroorotase purified from a cell-freeextract of E.coli in four steps has a turnover number of 297mol/min/(mol domain) for the conversion of L-dihydroorotateback to N-carbamyl-Laspartate with K₈ = 8.7 ± 1.5 µMfor L-dihydroorotate, a subunit molecular weight of 39 008 determinedfrom the sequence and 37 900 ± 400 when subjected toSDS–PAGE, and an isoelectric point of 5.7. Ultracentrifugalanalysis of the recombinant domain showed a single species ofs_20,w = 4.1 S and a single molecular species of M_r = 76 000corresponding to a dimer.     

Diphtheria toxin receptor binding domain substitution with interleukin-2: genetic construction and properties of a diphtheria toxin-related interleukin-2 fusion protein

We have genetically replaced the diphtheria toxin receptor bindingdomain with a synthetic gene encoding interleukin-2 (IL-2) anda translational stop signal. The diphtheria toxin-related T-cellgrowth factor fusion gene encodes a 70 586-d polypeptide, pro-BL-2-toxin.The mature form of IL-2- toxin has a deduced mol. wt of 68 086and is shown to be exported to the periplasmic compartment ofEscherichia coli (pABI508), and contain immunologic determinantsintrinsic to both its diphtheria toxin and IL-2 components.EL-2-toxin has been purified from periplasmic extracts of recombinantstrains of E.coli (pABI508) by immunoaffinity chromatographyusing immobilized anti-IL-2. The purified chimeric toxin isshown to selectively inhibit protein synthesis in IL-2 receptorbearing targeted cells, whereas cell lines which do not expressthe IL-2 receptor are resistant to IL-2-toxin action.     

Breakdown in the relationship between thermal and thermodynamic stability in an interleukin-1{beta} point mutant modified in a surface loop

Sequence variants of the ß-barrel protein interleukin-1ßhave been analyzed for their stabilities toward irreversiblethermal inactivation by monitoring the generation of light scatteringaggregates on heating. The derived temperatures for the onsetof aggregation (T_agg values) correlate well with the free energiesof unfolding of these proteins with the exception of one variant,Lys97—Val (K97V), which undergoes aggregation at a temperature7°C lower than expected based on its thermodynamic stability.This lower than expected thermal stability may be due to generationof an aggregation-prone unfolding intermediate at a temperaturelower than the T_m of the global transition. This hypothesisis supported by the location of residue 97 in the long 86–99loop which has structural features suggesting it may comprisea small, independent folding unit or microdomain. The excellentcorrelation of thermal and thermodynamic stabilities of sevenof the eight variants tested is consistent with accepted modelsfor thermal inactivation of proteins. At the same time the poorfit of the K97V variant underscores the risk in using thermalstability data in quantitative analysis of mutational studiesof the folding stability of proteins.     

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.     

Site-directed mutagenesis of Arg60 and Cys271 in ornithine transcarbamylase from rat liver

We have investigated the putative carbamylphosphate- and ornithine-bindingdomains in ornithine transcarbamylase from rat liver using site-directedmutagenesis. Arg60, present in the phosphate-binding motif X-Ser-X-Arg-Xand therefore implicated in the binding of the phosphate moietyof carbamylphosphate has been replaced with a leucine. Thisresults in a dramatic reduction of catalytic activity, althoughthe enzyme is synthesized in cells stably transfected with themutant clone and imported, correctly processed and assembledinto a homotrimer in mitochondria. The sole cysteine residue(Cys271) has been implicated in ornithine binding by the chemicalmodification studies of Marshall and Cohen in 1972 and 1980(J. Biol. Chem., 247, 1654–1668, 1669–1682; 255,7291–7295, 7296–7300). Replacement of this residuewith serine did not eliminate enzyme activity but affected theMichaelis constant for ornithine (K_b, increasing it 5-fold from0.71 to 3.7 mM and reduced the k_cat at pH 8.5 by 20-fold. Thesechanges represent a loss in apparent binding energy for theenzyme - ornithine complex of 2.9 kcal/mol, suggesting thatCys271 is normally involved in hydrogen bonding to the substrate,ornithine. The cysteine to serine substitution also caused thedissociation constant (Kä for the competitive inhibitor,L-norvaline to be increased 10-fold, from 12 to 120 µM.The small loss in binding energy and relatively high residualcatalytic activity of the mutant strongly suggests that a numberof other residues are involved in the binding of ornithine.The effect of replacement of Cys271 with serine was restrictedto the ornithine binding site of the enzyme since both the bindingconstant for carbamyl-phosphate (K_ia) and Michaelis constant(K_a) were not appreciably different for mutant and wild-typeenzymes. The pH optimum of the wild-type enzyme (8.6) is increasedto > 9.6 in the Ser271 mutant.     

Phage-enzymes: expression and affinity chromatography of functional alkaline phosphatase on the surface of bacteriophage

We have demonstrated that an active enzyme can be expressedon the surface of a bacteriophage. The gene encoding alkalinephosphatase from Escherichia coli was cloned upstream of gene3, which encodes a minor coat protein of the filamentous bacteriophage,fd. A fusion protein of the correct size was detected from viralparticles by Western blotting. Ultrafiltration confirmed thatthe enzyme fusion behaves as part of a larger structure as wouldbe expected of an enzyme fused to a viral particle. Both wild-typealkaline phosphatase (Argl66) and an active site mutant (Ala166) expressed in this way retain catalytic activity and havequalitatively similar kinetic properties to free enzyme. Valueswere obtained for K_m of 72.7 and 1070 µM respectivelywhilst relative k_cat for the mutant was 36% of that for wild-type.Phage particles expressing alkaline phosphatase were bound toan immobilized inhibitor (arsenate-Sepharose) and eluted withproduct (20 mM inorganic phosphate). In this way, the functionalenzyme is co-purified with the DNA encoding it. This may permita novel approach to enzyme engineering based on affinity chromatographyof mutant enzymes expressed on the phage surface.     

A structure-function relationship for the calcium affinities of regulatory proteins containing 'EF-hand' pairs

Using a series of homologous calcium-binding proteins, a quantitativestructure–activity relationship (QSAR), log(1/K_d) = –18.986– 1.6278(X₁) + 0.7981(X₂) + 0.2312(X₃), has been established,which relates the calcium-binding affinities (1/K_d) of the regulatoryproteins with (i) the net ligand charge (X₁) of the two calciumbinding loops, (ii) the hydrophobicity (X₂) of the ß-sheetsegment of the loops and (iii) the hydrophobicity (X₃) of thefour ‘EF-hand’ helices. It is found that the bindingaffinities are influenced by the ‘EF-hand’ pairrather than the individual ‘EF-hands’. The QSAR,in addition to explaining satisfactorily the large variationin the observed calcium affinities, can predict the affinitiesof the ‘EF-hand’ pairs in other proteins from theamino acid sequence and can also account for the changes inthe affinities caused by substitution in the hydrophobic and/ormetal-coordinating residues. Thus, this relationship can beemployed in protein design and engineering. The method is potentiallyuseful in the development of similar relationships for the bindingof other proteins to substrates, inhibitors, drugs and co-factors.     

Ex Vivo Expanded and Activated Natural Killer Cells Prolong the Overall Survival of Mice with Glioblastoma-like Cell-Derived Tumors

Glioblastoma (GBM) is the leading malignant intracranial tumor and is associated with a poor prognosis. Highly purified, activated natural killer (NK) cells, designated as genuine induced NK cells (GiNKs), represent a promising immunotherapy for GBM. We evaluated the anti-tumor effect of GiNKs in association with the programmed death 1(PD-1)/PD-ligand 1 (PD-L1) immune checkpoint pathway. We determined the level of PD-1 expression, a receptor known to down-regulate the immune response against malignancy, on GiNKs. PD-L1 expression on glioma cell lines (GBM-like cell line U87MG, and GBM cell line T98G) was also determined. To evaluate the anti-tumor activity of GiNKs in vivo, we used a xenograft model of subcutaneously implanted U87MG cells in immunocompromised NOG mice. The GiNKs expressed very low levels of PD-1. Although PD-L1 was expressed on U87MG and T98G cells, the expression levels were highly variable. Our xenograft model revealed that the retro-orbital administration of GiNKs and interleukin-2 (IL-2) prolonged the survival of NOG mice bearing subcutaneous U87MG-derived tumors. PD-1 blocking antibodies did not have an additive effect with GiNKs for prolonging survival. GiNKs may represent a promising cell-based immunotherapy for patients with GBM and are minimally affected by the PD-1/PD-L1 immune evasion axis in GBM.     

Expression and characterization of Lactobacillus 30a histidine decarboxylase in Escherichia coli

The genes coding for histidine decarboxylase from a wild-typestrain and an autoactivation mutant strain of Lactobacillus30a have been cloned and expressed in Escherichia coli. Themutant protein, G58D, has a single Asp for Gly substitutionat position 58. The cloned genes were placed under control ofthe ß-galactosidase promoter and the products arenatural length, not fusion proteins. The enzyme kinetics ofthe proteins isolated from E. coli are comparable to those isolatedfrom Lactobacillus 30a. At pH 4.8 the K_m of wild-type enzymeis 0.4 mM and the k_cat = 2800 min^–1; the correspondingvalues for G58D are 0.5 mM and 2750 min^–1. The wild-typeand G58D have autoactivation half-times of 21 and 9 h respectivelyunder pseudophysiological conditions of 150 mM K⁺ and pH 7.0.At pH 7.6 and 0.8 M K⁺ the half times are 4.9 and 2.9 h. Therelatively slow rate of autoactivation for purified proteinand the differences in cellular and non-cellular activationrates, coupled with the fact that wild-type protein is readilyactivated in wild-type Lactobacillus 30a but poorly activatedin E. coli, suggest that wild-type Lactobacillus 30a containsa factor, possibly an enzyme, that enhances the activation rate.     

The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment

The facile detection and purification of a recombinant proteinwithout detailed knowledge about its individual biochemicalproperties constitutes a problem of general interest in proteinengineering. The use of a novel kind of random peptide libraryfor the stepwise engineering of a C-terminal fusion peptidewhich confers binding activity towards streptavidin is describedin this study. Because of its widespread use as part of a varietyof conjugates and other affinity reagents, streptavidin constitutesthe binding partner of choice both for detection and purificationpurposes. The streptavidin-affinity tag was engineered at theC-terminus of the V_H domain as part of the D1.3 Fv fragmentwhich was functionally expressed in Escherichia coli. Irrespectiveof whether it was displayed by the V_H or the V_L domain, theoptimized version of the affinity peptide termed ‘Strep-tag’allowed the detection of the Fv fragment both on Western blotsand in ELISAs by a streptavidin–alkaline phosphatase conjugate.In addition, the one-step purification of the intact Fv fragmentcarrying a single Strep-tag at the C-terminus of only one ofits domains was achieved by affinity chromatography with streptavidin-agaroseusing very mild elution conditions.     

Phe496 and Leu497 are essential for receptor binding and cytotoxic action of the murine interleukin-4 receptor targeted fusion toxin DAB389-mIL-4

DAB₃₈₉-mIL-4 is a murine interleukin-4 (mIL-4) diphtheria toxin-relatedfusion protein which has been shown to be selectively toxicto cells expressing the mIL-4 receptor. In this report, we haveused site-directed and in-frame deletion mutagenesis to studythe role of the putative C-terminal -helix (helix E) of themIL-4 component of DAB₃₈₉-mIL-4 in the intoxication process.We demonstrate that deletion of the C-terminal 15 amino acidsof the fusion toxin leads to loss of cytotoxicity. The substitutionof Phe496 with either Pro, Ala or Tyr, results in a > 20-folddecrease in cytotoxic activity of the respective mutant fusiontoxins. In addition, substitution of Leu497 with either Alaor Glu results in a similar loss of cytotoxic activity. Allof these mutant forms of the mIL-4 fusion toxin demonstratea significant decrease in binding affinity (K_i) to the mIL-4receptor in a competitive radioligand binding assay. In markedcontrast, however, the substitution of Asp495 with Asn resultsin a 4-fold increase in cytotoxic potency and binding affinityto mIL-4 receptor bearing cells in vitro.     

Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module

Human c-Jun and c-Fos leucine zipper domains were examined fortheir ability to serve as autonomous dimerization domains aspart of a heterologous protein construct. Schistosoma japonicumglutathione S-transferase (GST) was fused to recombinant Junleucine zipper (rJunLZ) and Fos leucine zipper (rFosLZ) domains.SDS–PAGE ‘snapshot’ analyses based on disulphidelinkage of monomers demonstrated the ability of rJunLZ to functionas a dimerization motif in a foreign protein environment. Sterichindrance prevented formation of rJunLZ–GST::rFosLZ–GSTheterodimers whereas rJunLZ–GST::rFosLZ and rJunLZ::rFosLZ–GSTformed readily. Furthermore, rJunLZ–GST generated homodimerssuggesting fusion protein heterodimers interact differentlyto homodimers. Gel filtration chromatography confirmed thatGST is a dimer in solution and that attachment of a leucinezipper domain allows further interactions to take place. Sedimentationequilibrium analyses showed that GST is a stable dimer (K_a >10⁶ M^-1) with no higher multimeric forms. rFosLZ–GST weaklyassociates beyond a dimer (K_a {small tilde}4x10⁵ M^-1) and rJunLZ–GSTassociates indefinitely (K_a {small tilde}4x10⁶ M^-1), consistentwith an isodesmic model of association. The interaction of theseleucine zippers independently of GST association demonstratestheir utility in the modification of proteins when multimerformation is desired.