Orally administered L. lactis secreting an anti-TNF Nanobody demonstrate efficacy in chronic colitis.

Inflammatory bowel disease (IBD) is a chronic inflammatory gastrointestinal disorder. Systemic treatment of IBD patients with anti-tumor necrosis factor (TNF)-alpha antibodies has proven to be a highly promising approach, but several drawbacks remain, including side effects related to systemic administration and high cost of treatment. Lactococcus lactis was engineered to secrete monovalent and bivalent murine (m)TNF-neutralizing Nanobodies as therapeutic proteins. These therapeutic proteins are derived from fragments of heavy-chain camelid antibodies and are more stable than conventional antibodies. L. lactis-secreted anti-mTNF Nanobodies neutralized mTNF in vitro. Daily oral administration of Nanobody-secreting L. lactis resulted in local delivery of anti-mTNF Nanobodies at the colon and significantly reduced inflammation in mice with dextran sulfate sodium (DSS)-induced chronic colitis. In addition, this approach was also successful in improving established enterocolitis in interleukin 10 (IL10)(-/-) mice. Finally, L. lactis-secreted anti-mTNF Nanobodies did not interfere with systemic Salmonella infection in colitic IL10(-/-) mice.In conclusion, this report details a new therapeutic approach for treatment of chronic colitis, involving in situ secretion of anti-mTNF Nanobodies by orally administered L. lactis bacteria. Therapeutic application of these engineered bacteria could eventually lead to more effective and safer management of IBD in humans.

Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae.

Small, soluble single-domain fragments derived from the unique variable region of dromedary heavy-chain antibodies (VHHs) against enzymes are known to be potent inhibitors. The immunization of dromedaries with the TEM-1 and BcII beta-lactamases has lead to the isolation of such single-domain antibody fragments specifically recognizing and inhibiting those beta-lactamases. Two VHHs were isolated that inhibit TEM-1 and one BcII inhibiting VHH was identified. All inhibitory VHHs were tight-binding inhibitors. The 50% inhibitory concentrations were determined for all inhibitors and they were all in the same range as the enzyme concentration used in the assay. Addition of the VHHs to the TEM-1 beta-lactamase, expressed on the surface of bacteria, leads to a higher ampicillin sensitivity of the bacteria. This innovative strategy could generate multiple potent inhibitors for all types of beta-lactamases.

Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs.

Single-domain antibodies against various antigens are isolated from the unique heavy-chain antibodies of immunized camels and llamas. These minimal sized binders are very robust and bind the antigen with high affinity in a monomeric state. We evaluated the feasibility to produce soluble, functional bispecific and bivalent antibodies in Escherichia coli with camel single-domain antibody fragments as building blocks. Two single-domain antibody fragments were tethered by the structural upper hinge of a natural antibody to generate bispecific molecules. This linker was chosen for its protease resistance in serum and its natural flexibility to reorient the upstream and downstream located domains. The expression levels, ease of purification, and the solubility of the recombinant proteins were comparable with those of the constituent monomers. The individual moieties fully retain the binding capacity and the binding characteristics within the recombinant bispecific constructs. The easy generation steps and the biophysical properties of these bispecific and bivalent constructs based on camel single-domain antibody fragments makes them particularly attractive for use in therapeutic or diagnostic programs.

Unique single-domain antigen binding fragments derived from naturally occurring camel heavy-chain antibodies.

The humoral immune response of camels, dromedaries and llamas includes functional antibodies formed by two heavy chains and no light chains. The amino acid sequence of the variable domain of the naturally occurring heavy-chain antibodies reveals the necessary adaptations to compensate for the absence of the light chain. In contrast to the conventional antibodies, a large proportion of the heavy-chain antibodies acts as competitive enzyme inhibitors. Studies on the dromedary immunoglobulin genes start to shed light on the ontogeny of these heavy-chain antibodies. The presence of the heavy-chain antibodies and the possibility of immunizing a dromedary allows for the production of antigen binders consisting of a single domain only. These minimal antigen-binding fragments are well expressed in bacteria, bind the antigen with affinity in the nM range and are very stable. We expect that such camelid single domain antibodies will find their way into a number of biotechnological or medical applications. The structure of the camelid single domain is homologous to the human VH, however, the antigen-binding loop structures deviate fundamentally from the canonical structures described for human or mouse VHs. This has two additional advantages: (1) the camel or llama derived single domain antibodies might be an ideal scaffold for anti-idiotypic vaccinations; and (2) the development of smaller peptides or peptide mimetic drugs derived from of the antigen binding loops might be facilitated due to their less complex antigen binding site.

A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops.

BACKGROUND: Camelid serum contains a large fraction of functional heavy-chain antibodies - homodimers of heavy chains without light chains. The variable domains of these heavy-chain antibodies (VHH) have a long complementarity determining region 3 (CDR3) loop that compensates for the absence of the antigen-binding loops of the variable light chains (VL). In the case of the VHH fragment cAb-Lys3, part of the 24 amino acid long CDR3 loop protrudes from the antigen-binding surface and inserts into the active-site cleft of its antigen, rendering cAb-Lys3 a competitive enzyme inhibitor. RESULTS: A dromedary VHH with specificity for bovine RNase A, cAb-RN05, has a short CDR3 loop of 12 amino acids and is not a competitive enzyme inhibitor. The structure of the cAb-RN05-RNase A complex has been solved at 2.8 A. The VHH scaffold architecture is close to that of a human VH (variable heavy chain). The structure of the antigen-binding hypervariable 1 loop (H1) of both cAb-RN05 and cAb-Lys3 differ from the known canonical structures; in addition these H1 loops resemble each other. The CDR3 provides an antigen-binding surface and shields the face of the domain that interacts with VL in conventional antibodies. CONCLUSIONS: VHHs adopt the common immunoglobulin fold of variable domains, but the antigen-binding loops deviate from the predicted canonical structure. We define a new canonical structure for the H1 loop of immunoglobulins, with cAb-RN05 and cAb-Lys3 as reference structures. This new loop structure might also occur in human or mouse VH domains. Surprisingly, only two loops are involved in antigen recognition; the CDR2 does not participate. Nevertheless, the antigen binding occurs with nanomolar affinities because of a preferential usage of mainchain atoms for antigen interaction.

Potent enzyme inhibitors derived from dromedary heavy-chain antibodies.

Evidence is provided that dromedary heavy-chain antibodies, in vivo-matured in the absence of light chains, are a unique source of inhibitory antibodies. After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic alpha-amylase, it was demonstrated that a considerable amount of heavy-chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream. In contrast, the conventional antibodies apparently do not interact with the enzyme's active site. Next we illustrated that peripheral blood lymphocytes are suitable for one-step cloning of the variable domain fragments in a phage-display vector. By bio-panning, several antigen-specific single-domain fragments are readily isolated for both enzymes. In addition we show that among those isolated fragments active site binders are well represented. When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays. The low complexity of the antigen-binding site of these single-domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors.

The integrin alpha 2 beta 1 (GPIa/IIa)-I-domain inhibits platelet-collagen interaction.

The integrin alpha 2 beta 1 is a major cellular receptor for collagen. The alpha 2 subunit contains an +/- 200 amino acids inserted domain (I-domain) in the N-terminal region. A certain degree of homology exists between the I-domains found in integrins, collagen and the A-domains of vWF. The alpha 2-I-domain encoding region (aa residues D145 to S334) was obtained by RT-PCR from mRNA of non stimulated human PBL's. The primers were designed to introduce the necessary restriction sites for cloning of the DNA fragment in frame downstream of the malE gene, as well as a stop codon after the last triplet. The resulting construct pMAL-c2-alpha 2-I allows the expression of the I-domain, fused to the C-terminus of maltose binding protein (mal). The alpha 2-I-mal is purified from the bacterial extract by affinity chromatography on an amylose column. The purified alpha 2-I-mal has been characterized by ELISA's. The alpha 2-I-mal bound to immobilised collagen type I in a concentration dependent manner and could be blocked by the functional monoclonal anti-alpha 2 beta 1 antibody 6F1. The interaction of alpha 2-I-mal with collagen furthermore is Mg(2+)-dependent since the binding was inhibited in the presence of 10 mM EDTA or 10 mM Ca2+ but sustained in the presence of 10 mM Mg2+. Finally, alpha 2-I-mal itself was able to inhibit adhesion of washed platelets to collagen immobilised on a microtiterplate in a dose-dependent manner (alpha 2-I-mal IC50:0.7 microM) as well as platelet aggregation induced by collagen type I (alpha 2-I-mal IC50:0.7 microM). With these results we could confirm that the alpha 2-I-domain represents the collagen-binding site of alpha 2 beta 1 and we furthermore could indicate that this domain is able to prevent platelet adhesion to collagen and collagen-induced platelet aggregation, pointing to the primordial role of alpha 2-I-mal and hence of alpha 2 beta 1 in platelet-collagen interaction.

Anticoagulant repertoire of the hookworm Ancylostoma caninum.

Hookworms are hematophagous nematodes that infect a wide range of mammalian hosts, including humans. There has been speculation for nearly a century as to the identity of the anticoagulant substances) used by these organisms to subvert host hemostasis. Using molecular cloning, we describe a family of potent small protein (75-84 amino acids) anticoagulants from the hookworm Ancylostoma caninum termed AcAP (A. caninum anticoagulant protein). Two recombinant AcAP members (AcAP5 and AcAP6) directly inhibited the catalytic activity of blood coagulation factor Xa (fXa), while a third form (AcAPc2) predominantly inhibited the catalytic activity of a complex composed of blood coagulation factor VIIa and tissue factor (fVIIa/TF). The inhibition of fVIIa/TF was by a unique mechanism that required the initial formation of a binary complex of the inhibitor with fXa at a site on the enzyme that is distinct from the catalytic center (exo-site). The sequence of AcAPc2 as well as the utilization of an exo-site on fXa distinguishes this inhibitor from the mammalian anticoagulant TFPI (tissue factor pathway inhibitor), which is functionally equivalent with respect to fXa-dependent inhibition of fIIa/TF. The relative sequence positions of the reactive site residues determined for AcAP5 with the homologous regions in AcAP6 and AcAPc2 as well as the pattern of 10 cysteine residues present in each of the inhibitors suggest that the AcAPs are distantly related to the family of small protein serine protease inhibitors found in the nonhematophagous nematode Ascaris lumbricoides var. suum.

Surface expression and ligand-based selection of cDNAs fused to filamentous phage gene VI.

We describe a novel phage display system that affords the surface expression and hence affinity selection of cDNAs. The strategy is based on a new approach to functionally display proteins on filamentous phage through the attachment to the C-terminus of the minor coat protein VI. The utility of the method was evaluated using a cDNA library derived from the parasite Ancylostoma caninum. cDNA sequences were fused in each of the three reading frames to the 3'-end of the M13 gene VI expressed by a phagemid vector. Phages rescued from this cDNA expression library were subjected to biopanning against two serine proteases, trypsin and the human coagulation factor Xa. This led to the identification of cDNAs encoding novel members of two different families of serine protease inhibitors. The authenticity of the cDNA selected with trypsin as the target was demonstrated by purifying the encoded potent Kunitz-type inhibitor from an Ancylostoma caninum extract. The rapid isolation of specific cDNAs with the protein VI monovalent display system should facilitate the search for novel biologically important ligands.

High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris.

Tick anticoagulant peptide (TAP) is a potent and specific inhibitor of the blood coagulation protease Factor Xa. We designed and assembled a synthetic TAP-encoding gene (tapo) based on codons preferentially observed in the highly expressed Pichia pastoris alcohol oxidase 1 gene (AOX1), and fused it to a novel hybrid secretory prepro leader sequence. Expression from this gene yielded biologically active rTAP, which was correctly processed at the amino-terminal fusion site, and accumulated in the medium to approximately 1.7 g/l. This corresponds to a molar concentration of 0.24 mM, and is the highest yet described for a recombinant product secreted from P. pastoris. It also represents a seven-fold improvement in productivity compared to rTAP secretion from Saccharomyces cerevisiae, making P. pastoris an attractive host for the industrial-scale production of this potential therapeutic agent. This system was also used to prepare 21 mg 15N-rTAP, 11 mg 13C-rTAP and 27 mg 15N/13C-rTAP, with isotope incorporation levels higher than 98%, and purities sufficient to allow their use in determining the solution structure of the tick anticoagulant peptide using high field NMR.

An echistatin-like Arg-Gly-Asp (RGD)-containing sequence in the heavy chain CDR3 of a murine monoclonal antibody that inhibits human platelet glycoprotein IIb/IIIa function.

We describe the production and biochemical characterization of the first GPIIb/IIIa-inhibiting monoclonal antibody that contains an RGD sequence in the CDR3 region of the heavy chain. Monoclonal antibodies obtained by immunizing mice with human platelets were screened using consecutive ELISAs based on human platelets and immuno-affinity-purified glycoprotein (GP) IIb/IIIa coated on microtitre plates. Out of 30 monoclonal antibodies reacting with GPIIb/IIIa, one, MA-16N7C2, potently inhibited platelet aggregation induced by ADP, thrombin, arachidonic acid, collagen, U46619, adrenaline and platelet-activating factor, whereas ristocetin-induced aggregation was unaffected. MA-16N7C2 (IgG2a) bound approximately 4 times faster to activated than to resting platelets, with a Kdcalc of 6.6nM and of 17.5nM, respectively. Equilibrium binding studies to non-activated platelets showed a Kd of 18.2nM with 41 x 10(3) binding sites per platelet. The antibody recognized GPIIb/IIIa only as a Ca(2+)-dependent complex. MA-16N7C2 blocked fibrinogen and von Willebrand factor binding to GPIIb/IIIa in a competitive manner with a Ki of 8.5nM and 13.2nM, respectively. Sequence analysis revealed a RGD-containing sequence with homology to disintegrins, in the CDR3 region of the heavy chain. That this RGD-containing sequence could be involved in the interaction of the antibody to GPIIb/IIIa was finally indicated by showing that the binding is completely and competitively inhibited by echistatin.

Engineering cysteine mutants to obtain crystallographic phases with a cutinase from Fusarium solani pisi.

Cutinases are extracellular enzymes involved in the disruption of cutine, an insoluble polyester which covers the surface of plants. They belong to a class of serine esterases that are able to hydrolyse fatty acid esters and emulsified triglycerides as efficiently as lipases, but without displaying interfacial activation. Classical crystallographic methods for obtaining heavy-atom derivatives failed, so the cutinase structure has been solved exclusively by the multiple isomorphous replacement method using four Hg derivatives obtained from mutants S4C, S92C, S120C and S129C. Two of these derivatives behaved as expected: (i) the cys mutant of the catalytic Ser S120C, located at the surface of the active site pocket, leads to a good derivative; and (ii) the Hg atom of the derivative obtained with the S92C mutant is completely accessible to the solvent and occupies two alternative positions--consequently a poor derivative results. In contrast, two mutants show an unexpected behaviour: (i) the Hg atom in the S129C mutant was completely buried 10 A below the protein surface and yielded the best derivative; and (ii) a poor quality derivative was obtained with the S4C mutant. Cys 4 belongs to the disordered propeptide 1-16. The Cys 4 bound Hg atom is located in front of the Asp58 side chain, but neither Cys4 nor parts of the propeptide are clearly visible in the electron density maps of the derivative structure.

Biochemical characterization of single-chain chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody and single-chain urokinase.

K12G0S32 is a 57-kDa recombinant single-chain chimeric plasminogen activator consisting of scFv-K12Go, a single-chain variable-region antigen-binding fragment (Fv) of the monoclonal antibody MA-15C5, which is specific for fragment D-dimer of human cross-linked fibrin, and a low-molecular-mass (33 kDa) urokinase-type plasminogen activator (u-PA-33k) containing amino acids Ala132-Leu411 (Holvoet, P., Laroche, Y., Lijnen, H. R., Van Cauwenberghe, R., Demarsin, E., Brouwers, E., Matthyssens, G. & Collen D. (1991) J. Biol. Chem. 266, 19717-19724). In addition, the Arg156-Phe157 thrombin-cleavage site in the u-PA moiety of K12G0S32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K12G0S32, determined in a system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated plasma, was found to be only twofold higher than that of intact single-chain u-Pa (rscu-PA), but 17-fold higher than that of rscu-PA(M), a variant of rscu-PA in which the thrombin-cleavage site was removed by substitution of Phe157 with Asp. The fibrinolytic potency of K12G0S32T, with an intact thrombin-cleavage site, was 6-15-fold higher than that of rscu-PA. Conversion of 1 microM single-chain K12G0S32 or rscu-PA(M) into their two-chain derivatives with plasmin occurred at a rate of 1.0 +/- 0.15 nmol.min-1.nmol plasmin-1 and 0.85 +/- 0.074 nmol.min-1.nmol plasmin-1, compared to 14 +/- 2.3 nmol.min-1.nmol plasmin-1 and 18 +/- 2.6 nM.min-1.nmol plasmin-1 for K12G0S32T and rscu-PA, respectively. Purified fragment D-dimer of human cross-linked fibrin inhibited the fibrinolytic potency of single-chain K12G0S32T, but not of two-chain K12G0S32T, in a dose-dependent manner. Furthermore, the fibrinolytic potencies of two-chain K12G0S32 and K12G0S32T were not significantly higher than those of recombinant two-chain u-PA (rtcu-PA) or of rtcu-PA(M). These findings suggest that the 59-fold increase in fibrinolytic potency of K12G0S32T, relative to that of rscu-PA(M), is due both to targeting of the activator to the clot via the single-chain Fv fragment (sixfold increase) and to a more efficient conversion of single-chain K12G0S32T to its two-chain derivative (eightfold increase). Thus, targeting to clots by means of fibrin-specific antibodies results in a significant increase of the fibrinolytic potency of single-chain but not of two-chain u-PA.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 1. Crystallography and site-directed mutagenesis of metal binding sites.

The structure and function of the xylose (glucose) isomerase from Actinoplanes missouriensis have been analyzed by X-ray crystallography and site-directed mutagenesis after cloning and overexpression in Escherichia coli. The crystal structure of wild-type enzyme has been refined to an R factor of 15.2% against diffraction data to 2.2-A resolution. The structures of a number of binary and ternary complexes involving wild-type and mutant enzymes, the divalent cations Mg2+, Co2+, or Mn2+, and either the substrate xylose or substrate analogs have also been determined and refined to comparable R factors. Two metal sites are identified. Metal site 1 is four-coordinated and tetrahedral in the absence of substrate and is six-coordinated and octahedral in its presence; the O2 and O4 atoms of linear inhibitors and substrate bind to metal 1. Metal site 2 is octahedral in all cases; its position changes by 0.7 A when it binds O1 of the substrate and by more than 1 A when it also binds O2; these bonds replace bonds to carboxylate ligands from the protein. Side chains involved in metal binding have been substituted by site-directed mutagenesis. The biochemical properties of the mutant enzymes are presented. Together with structural data, they demonstrate that the two metal ions play an essential part in binding substrates, in stabilizing their open form, and in catalyzing hydride transfer between the C1 and C2 positions.

Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 2. Site-directed mutagenesis of the xylose binding site.

Site-directed mutagenesis in the active site of xylose isomerase derived from Actinoplanes missouriensis is used to investigate the structural and functional role of specific residues. The mutagenesis work together with the crystallographic studies presented in detail in two accompanying papers adds significantly to the understanding of the catalytic mechanism of this enzyme. Changes caused by introduced mutations emphasize the correlation between substrate specificity and cation preference. Mutations in both His 220 and His 54 mainly affect the catalytic rate constant, with catalysis being severely reduced but not abolished, suggesting that both histidines are important, but not essential, for catalysis. Our results thus challenge the hypothesis that His 54 acts as an obligatory catalytic base for ring opening; this residue appears instead to be implicated in governing the anomeric specificity. With none of the active site histidines acting as a catalytic base, the role of the cations in catalyzing proton transfer is confirmed. In addition, Lys 183 appears to play a crucial part in the isomerization step, by assisting the proton shuttle. Other residues also are important but to a lesser extent. The conserved Lys 294 is indirectly involved in binding the activating cations. Among the active site aromatic residues, the tryptophans (16 and 137) play a role in maintaining the general architecture of the substrate binding site while the role of Phe 26 seems to be purely structural.

Fusarium solani cutinase is a lipolytic enzyme with a catalytic serine accessible to solvent.

Lipases belong to a class of esterases whose activity on triglycerides is greatly enhanced at lipid-water interfaces. This phenomenon, called interfacial activation, has a structural explanation: a hydrophobic lid, which at rest covers the catalytic site, is displaced on substrate or inhibitor binding and probably interacts with the lipid matrix. Fusarium solani pisi cutinase belongs to a group of homologous enzymes of relative molecular mass 22-25K (ref. 7) capable of degrading cutin, the insoluble lipid-polyester matrix covering the surface of plants, and hydrolysing triglycerides. Cutinases differ from classical lipases in that they do not exhibit interfacial activation; they are active on soluble as well as on emulsified triglycerides. Cutinases therefore establish a bridge between esterases and lipases. We report here the three-dimensional structure of a recombinant cutinase from F. solani pisi, expressed in Escherichia coli. Cutinase is an alpha-beta protein; the active site is composed of the triad Ser 120, His 188 and Asp 175. Unlike other lipases, the catalytic serine is not buried under surface loops, but is accessible to solvent. This could explain why cutinase does not display interfacial activation.

Characterization of phosphinothricin acetyltransferase and C-terminal enzymatically active fusion proteins.

Enhanced expression of the bialaphos resistance (bar) from Streptomyces hygroscopicus, which confers resistance to the herbicides bialaphos and phosphinothricin (PPT), has been obtained in Escherichia coli using a vector system based on translational coupling. The gene product, PPT acetyltransferase, was purified to homogeneity and its enzymatic properties were analyzed. Hybrid gene constructs with gene fragments fused to the 3'-terminus of bar yield fusion proteins having acetyltransferase activity, with a Michaelis constant for the PPT substrate comparable to the unmodified enzyme. The bar gene represents a selectable and assayable reporter gene especially suitable for 3'-terminal gene fusions.

Two structural domains as a general fold of the toxic fragment of the Bacillus thuringiensis delta-endotoxins.

The unfolding by guanidine hydrochloride of the toxic fragment of a Bacillus thuringiensis toxin belonging to the CryIC class reveals a two-step denaturation under both acid and alkaline conditions. This demonstrates the existence of two structural domains as building blocks for this toxin. Protease digests performed on a CryIA(b) and CryIC B. thuringiensis toxin, under native and partially denatured conditions, confirm this conclusion. Whereas the native CryIC toxin is completely protease resistant, the CryIA(b) toxin, earlier described as consisting of two structural domains [Convents, D., Houssier, C., Lasters, I. & Lauwereys, M. (1990) J. Biol. Chem. 265, 1369-1375], is cleaved by three proteases, resulting in at least two common fragments. This suggests that this toxin is built up of two globular units linked by a protease-susceptible linker. The detection of a stable intermediate along the denaturation curve allows us to study and compare the consecutive unfolding of the structural domains for both toxins. By addition of a protease, under conditions where such an unfolding intermediate exists, a single denaturation phase can be assigned to a specific part of the protein. These experiments lead to the conclusion that the domain whose stability is highly dependent on pH corresponds to the N-terminal half of both toxins.

Crystallization and preliminary X-ray study of a recombinant cutinase from Fusarium solani pisi.

Recombinant cutinase from Fusarium solani pisi is expressed and excreted with very high yields in Escherichia coli cultures. Cutinase was crystallized at 20 degrees C using the vapour diffusion technique, with polyethylene glycol 6000 as precipitant. Best crystals were obtained at pH 7.0 with polyethylene glycol 6000 as precipitant. Best crystals were obtained at pH 7.0 with polyethylene glycol at 15 to 20%. They are monoclinic, with space group P2(1) and cell dimensions a = 35.1 A, b = 67.4 A, c = 37.05 A and beta = 94.0 degrees; they diffract beyond 1.5 A resolution. The asymmetric unit contains one molecule of 22,000 Da (Vm = 1.98 A3/Da; 38% water).

The Bacillus thuringiensis delta-endotoxin. Evidence for a two domain structure of the minimal toxic fragment.

The conformational characteristics of the minimal toxic fragment of the delta-endotoxin from Bacillus thuringiensis berliner 1715 were examined by fluorescence and circular dichroism spectroscopy. This insecticidal protein, specifically toxic to lepidopteran species, was found to consist of two structural domains. Experimental evidence for this conclusion was provided by biphasic guanidine hydrochloride unfolding curves at different pH values and electrophoretic patterns of protease digests. Two stable fragments of comparable molecular weight were obtained using four different broad specificity proteolytic enzymes. A secondary structure model was constructed using seven B. thuringiensis toxin sequences. These toxins were selected on the basis of their limited sequence homology and represent all known insecticidal specificities. Despite this divergence, a consensus secondary structure pattern was obtained, confirming the structural homology among the toxins. The N-terminal halves of all toxins are predicted to be relatively rich in alpha-helix structure and the C-terminal parts to contain alternating beta-strand and coil structures. The latter seems characteristic for a beta-sheet conformation. Comparing this model to the unfolding data obtained by circular dichroism, whose far UV signal gives a measure of the alpha-helix content, allowed us to delineate the structural domains into the primary structure.