"Affimer" synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation.

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol, and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidized low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low-density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified five synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently labeled oxLDL by the LOX-1-expressing cell model was inhibited with subnanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p < 0.01) by preincubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease.

Rapid Quantification of C. difficile Glutamate Dehydrogenase and Toxin B (TcdB) with a NanoBiT Split-Luciferase Assay.

C. difficile infection (CDI) is a leading healthcare-associated infection with a high morbidity and mortality and is a financial burden. No current standalone point-of-care test (POCT) is sufficient for the identification of true CDI over a disease-free carriage of C. difficile, so one is urgently required to ensure timely, appropriate treatment. Here, two types of binding proteins, Affimers and nanobodies, targeting two C. difficile biomarkers, glutamate dehydrogenase (GDH) and toxin B (TcdB), are combined in NanoBiT (NanoLuc Binary Technology) split-luciferase assays. The assays were optimized and their performance controlling parameters were examined. The 44 fM limit of detection (LoD), 4-5 log range and 1300-fold signal gain of the TcdB assay in buffer is the best observed for a NanoBiT assay to date. In the stool sample matrix, the GDH and TcdB assay sensitivity (LoD = 4.5 and 2 pM, respectively) and time to result (32 min) are similar to a current, commercial lateral flow POCT, but the NanoBit assay has no wash steps, detects clinically relevant TcdB over TcdA, and is quantitative. Development of the assay into a POCT may drive sensitivity further and offer an urgently needed ultrasensitive TcdB test for the rapid diagnosis of true CDI. The NanoBiTBiP (NanoBiT with Binding Proteins) system offers advantages over NanoBiT assays with antibodies as binding elements in terms of ease of production and assay performance. We expect this methodology and approach to be generally applicable to other biomarkers.

Affimer proteins as a tool to modulate fibrinolysis, stabilize the blood clot, and reduce bleeding complications.

Bleeding complications secondary to surgery, trauma, or coagulation disorders are important causes of morbidity and mortality. Although fibrin sealants are considered to minimize blood loss, this is not widely adopted because of its high cost and/or risk for infection. We present a novel methodology employing nonantibody fibrinogen-binding proteins, termed Affimers, to stabilize fibrin networks with the potential to control excessive bleeding. Two fibrinogen-specific Affimer proteins, F5 and G2, were identified and characterized for their effects on clot structure/fibrinolysis, using turbidimetric and permeation analyses and confocal and electron microscopy. Binding studies and molecular modeling identified interaction sites, whereas plasmin generation assays determined effects on plasminogen activation. In human plasma, F5 and G2 prolonged clot lysis time from 9.8 ± 1.1 minutes in the absence of Affimers to 172.6 ± 7.4 and more than 180 minutes (P < .0001), respectively, and from 7.6 ± 0.2 to 28.7 ± 5.8 (P < .05) and 149.3 ± 9.7 (P < .0001) minutes in clots made from purified fibrinogen. Prolongation in fibrinolysis was consistent across plasma samples from healthy control patients and individuals at high bleeding risk. F5 and G2 had a differential effect on clot structure and G2 profoundly altered fibrin fiber arrangement, whereas F5 maintained physiological clot structure. Affimer F5 reduced fibrin-dependent plasmin generation and was predicted to bind fibrinogen D fragment close to tissue plasminogen activator (tPA; residues γ312-324) and plasminogen (α148-160) binding sites, thus interfering with tPA-plasminogen interaction and representing 1 potential mechanism for modulation of fibrinolysis. Our Affimer proteins provide a novel methodology for stabilizing fibrin networks with potential future clinical implications to reduce bleeding risk.

Fibrinogen interaction with complement C3: a potential therapeutic target to reduce thrombosis risk.

Complement C3 binds fibrinogen and compromises fibrin clot lysis thereby enhancing thrombosis risk. We investigated the role of fibrinogen-C3 interaction as a novel therapeutic target to reduce thrombosis risk by analysing: i) consistency in the fibrinolytic properties of C3, ii) binding sites between fibrinogen and C3 and iii) modulation of fibrin clot lysis by manipulating fibrinogen-C3 interactions. Purified fibrinogen and C3 from the same individuals (n=24) were used to assess inter-individual variability in the anti-fibrinolytic effects of C3. Microarray screening and molecular modelling evaluated C3 and fibrinogen interaction sites. Novel synthetic conformational proteins, termed Affimers, were used to modulate C3-fibrinogen interaction and fibrinolysis. C3 purified from patients with type 1 diabetes showed enhanced prolongation of fibrinolysis compared with healthy control protein [195±105 and 522±166 seconds, respectively (p=0.04)], with consistent effects but a wider range (5-51% and 5-18% lysis prolongation, respectively). Peptide microarray screening identified 2 potential C3-fibrinogen interactions sites within fibrinogen ß chain (residues 424-433, 435-445). One fibrinogen-binding Affimer was isolated that displayed sequence identity with C3 in an exposed area of the protein. This Affimer abolished C3-induced prolongation of fibrinolysis (728±25.1 seconds to 632±23.7 seconds, p=0.005) and showed binding to fibrinogen in the same region that is involved in C3-fibrinogen interactions. Moreover, it shortened plasma clot lysis of patients with diabetes, cardiovascular disease or controls by 7-11%. C3 binds fibrinogen ß-chain and disruption of fibrinogen-C3 interaction using Affimer proteins enhances fibrinolysis, which represents a potential novel target tool to reduce thrombosis in high risk individuals.

Affimer proteins inhibit immune complex binding to FcγRIIIa with high specificity through competitive and allosteric modes of action.

Protein-protein interactions are essential for the control of cellular functions and are critical for regulation of the immune system. One example is the binding of Fc regions of IgG to the Fc gamma receptors (FcγRs). High sequence identity (98%) between the genes encoding FcγRIIIa (expressed on macrophages and natural killer cells) and FcγRIIIb (expressed on neutrophils) has prevented the development of monospecific agents against these therapeutic targets. We now report the identification of FcγRIIIa-specific artificial binding proteins called "Affimer" that block IgG binding and abrogate FcγRIIIa-mediated downstream effector functions in macrophages, namely TNF release and phagocytosis. Cocrystal structures and molecular dynamics simulations have revealed the structural basis of this specificity for two Affimer proteins: One binds directly to the Fc binding site, whereas the other acts allosterically.

C-Terminal Domain of the Human Zinc Transporter hZnT8 Is Structurally Indistinguishable from Its Disease Risk Variant (R325W).

The human zinc transporter 8 (hZnT8) plays important roles in the storage of insulin in the secretory vesicles of pancreatic ß cells. hZnT8 consists of a transmembrane domain, with its N- and C-termini protruding into the cytoplasm. Interestingly, the exchange of arginine to tryptophan at position 325 in the C-terminal domain (CTD) increases the risk of developing type 2 diabetes mellitus (T2D). In the present study, the CTDs of hZnT8 (the wild-type (WT) and its disease risk variant (R325W)) were expressed, purified, and characterized in their native forms by biophysical techniques. The data reveal that the CTDs form tetramers which are stabilized by zinc binding, and exhibit negligible differences in their secondary structure content and zinc-binding affinities in solution. These findings provide the basis for conducting further structural studies aimed at unravelling the molecular mechanism underlying the increased susceptibility to develop T2D, which is modulated by the disease risk variant.

Oxygen Activation Switch in the Copper Amine Oxidase of Escherichia coli.

Copper amine oxidases (CuAOs) are metalloenzymes that reduce molecular oxygen to hydrogen peroxide during catalytic turnover of primary amines. In addition to Cu2+ in the active site, two peripheral calcium sites, ∼32 Šfrom the active site, have roles in Escherichia coli amine oxidase (ECAO). The buried Ca2+ (Asp533, Leu534, Asp535, Asp678, and Ala679) is essential for full-length protein production, while the surface Ca2+ (Glu573, Tyr667, Asp670, and Glu672) modulates biogenesis of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor. The E573Q mutation at the surface site prevents calcium binding and TPQ biogenesis. However, TPQ biogenesis can be restored by a suppressor mutation (I342F) in the proposed oxygen delivery channel to the active site. While supporting TPQ biogenesis (∼60% WTECAO TPQ), I342F/E573Q has almost no amine oxidase activity (∼4.6% WTECAO activity). To understand how these long-range mutations have major effects on TPQ biogenesis and catalysis, we employed ultraviolet-visible spectroscopy, steady-state kinetics, inhibition assays, and X-ray crystallography. We show that the surface metal site controls the equilibrium (disproportionation) of the Cu2+-substrate reduced TPQ (TPQAMQ) Cu+-TPQ semiquinone (TPQSQ) couple. Removal of the calcium ion from this site by chelation or mutagenesis shifts the equilibrium to Cu2+-TPQAMQ or destabilizes Cu+-TPQSQ. Crystal structure analysis shows that TPQ biogenesis is stalled at deprotonation in the Cu2+-tyrosinate state. Our findings support WTECAO using the inner sphere electron transfer mechanism for oxygen reduction during catalysis, and while a Cu+-tyrosyl radical intermediate is not essential for TPQ biogenesis, it is required for efficient biogenesis.

Passive Picoinjection Enables Controlled Crystallization in a Droplet Microfluidic Device.

Segmented flow microfluidic devices offer an attractive means of studying crystallization processes. However, while they are widely employed for protein crystallization, there are few examples of their use for sparingly soluble compounds due to problems with rapid device fouling and irreproducibility over longer run-times. This article presents a microfluidic device which overcomes these issues, as this is constructed around a novel design of "picoinjector" that facilitates direct injection into flowing droplets. Exploiting a Venturi junction to reduce the pressure within the droplet, it is shown that passive injection of solution from a side-capillary can be achieved in the absence of an applied electric field. The operation of this device is demonstrated for calcium carbonate, where highly reproducible results are obtained over long run-times at high supersaturations. This compares with conventional devices that use a Y-junction to achieve solution loading, where in-channel precipitation of calcium carbonate occurs even at low supersaturations. This work not only opens the door to the use of microfluidics to study the crystallization of low solubility compounds, but the simple design of a passive picoinjector will find wide utility in areas including multistep reactions and investigation of reaction dynamics.

Exploiting orientation-selective DEER: determining molecular structure in systems containing Cu(ii) centres.

Orientation-selective DEER (Double Electron-Electron Resonance) measurements were conducted on a series of rigid and flexible molecules containing Cu(ii) ions. A system with two rigidly held Cu(ii) ions was afforded by the protein homo-dimer of copper amine oxidase from Arthrobacter globiformis. This system provided experimental DEER data between two Cu(ii) ions with a well-defined distance and relative orientation to assess the accuracy of the methodology. Evaluation of orientation-selective DEER (os DEER) on systems with limited flexibility was probed using a series of porphyrin-based Cu(ii)-nitroxide and Cu(ii)-Cu(ii) model systems of well-defined lengths synthesized for this project. Density functional theory was employed to generate molecular models of the conformers for each porphyrin-based Cu(ii) dimer studied. Excellent agreement was found between DEER traces simulated using these computed conformers and the experimental data. The performance of different parameterised structural models in simulating the experimental DEER data was also investigated. The results of this analysis demonstrate the degree to which the DEER data define the relative orientation of the two Cu(ii) ions and highlight the need to choose a parameterised model that captures the essential features of the flexibility (rotational freedom) of the system being studied.

Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2.

PepT1 and PepT2 are major facilitator superfamily (MFS) transporters that utilize a proton gradient to drive the uptake of di- and tri-peptides in the small intestine and kidney, respectively. They are the major routes by which we absorb dietary nitrogen and many orally administered drugs. Here, we present the crystal structure of PepT(So), a functionally similar prokaryotic homologue of the mammalian peptide transporters from Shewanella oneidensis. This structure, refined using data up to 3.6 Å resolution, reveals a ligand-bound occluded state for the MFS and provides new insights into a general transport mechanism. We have located the peptide-binding site in a central hydrophilic cavity, which occludes a bound ligand from both sides of the membrane. Residues thought to be involved in proton coupling have also been identified near the extracellular gate of the cavity. Based on these findings and associated kinetic data, we propose that PepT(So) represents a sound model system for understanding mammalian peptide transport as catalysed by PepT1 and PepT2.

Probing the molecular mechanisms in copper amine oxidases by generating heterodimers.

For some homodimeric copper amine oxidases (CuAO), there is suggestive evidence of differential activity at the two active sites implying potential cooperativity between the two monomers. To examine this phenomenon for the Arthrobacter globiformis CuAO (AGAO), we purified a heterodimeric form of the enzyme for comparison with the homodimer. The heterodimer comprises an active wild-type monomer and an inactive monomer in which an active-site tyrosine is mutated to phenylalanine (Y382F). This mutation prevents the formation of the trihydroxyphenylalanine quinone (TPQ) cofactor. A pETDuet vector and a dual fusion tag strategy was used to purify heterodimers (WT/Y382F) from homodimers. Purity was confirmed by western blot and native PAGE analyses. Spectral and kinetic studies support the view that whether there are one or two functional monomers in the dimer, the properties of each functional monomer are the same, thus indicating no communication between the active sites in this bacterial enzyme.

Targeting Grb2 SH3 Domains with Affimer Proteins Provides Novel Insights into Ras Signalling Modulation.

Src homology 3 (SH3) domains play a critical role in mediating protein-protein interactions (PPIs) involved in cell proliferation, migration, and the cytoskeleton. Despite their abundance in the human proteome, the functions and molecular interactions of many SH3 domains remain unknown, and this is in part due to the lack of SH3-domain-specific reagents available for their study. Affimer proteins have been developed as affinity reagents targeting a diverse range of targets, including those involved in PPIs. In this study, Affimer proteins were isolated against both the N- and C-terminal SH3 domains (NSH3 and CSH3) of growth-factor-receptor-bound protein 2 (Grb2), an adapter protein that provides a critical link between cell surface receptors and Ras signalling pathways. Targeting the CSH3 alone for the inhibition of PPIs appeared sufficient for curtailing Ras signalling in mammalian cell lines stimulated with human epidermal growth factor (EGF), which conflicts with the notion that the predominant interactions with Ras activating Son of sevenless (SOS) occur via the NSH3 domain. This result supports a model in which allosteric mechanisms involved in Grb2-SOS1 interaction modulate Ras activation.

Anti-TNF Thioester Glucocorticoid Antibody-Drug Conjugate Fully Inhibits Inflammation with Minimal Effect on Systemic Corticosterone Levels in a Mouse Arthritis Model.

We describe the discovery of a thioester-containing glucocorticoid receptor modulator (GRM) payload and the corresponding antibody-drug conjugate (ADC). Payload 6 was designed for rapid hepatic inactivation to minimize systemic exposure of nonconjugated GRM. Mouse PK indicated that 6 is cleared 10-fold more rapidly than a first-generation GRM payload, resulting in 10-fold lower exposure and 3-fold decrease in Cmax. The anti-mTNF conjugate ADC5 fully inhibited inflammation in mouse contact hypersensitivity with minimal effects on corticosterone, a biomarker for systemic GRM effects, at doses up to and including 100 mg/kg. Concomitant inhibition of P1NP suggests potential delivery to cells involved in the remodeling of bone, which may be a consequence of TNF-targeting or bystander payload effects. Furthermore, ADC5 fully suppressed inflammation in collagen-induced arthritis mouse model after one 10 mg/kg dose for 21 days. The properties of the anti-hTNF conjugate were suitable for liquid formulation and may enable subcutaneous dosing.

An anti-TNF-glucocorticoid receptor modulator antibody-drug conjugate is efficacious against immune-mediated inflammatory diseases.

Glucocorticoids (GCs) are efficacious drugs used for treating many inflammatory diseases, but the dose and duration of administration are limited because of severe side effects. We therefore sought to identify an approach to selectively target GCs to inflamed tissue. Previous work identified that anti-tumor necrosis factor (TNF) antibodies that bind to transmembrane TNF undergo internalization; therefore, an anti-TNF antibody-drug conjugate (ADC) would be mechanistically similar, where lysosomal catabolism could release a GC receptor modulator (GRM) payload to dampen immune cell activity. Consequently, we have generated an anti-TNF-GRM ADC with the aim of inhibiting pro-inflammatory cytokine production from stimulated human immune cells. In an acute mouse model of contact hypersensitivity, a murine surrogate anti-TNF-GRM ADC inhibited inflammatory responses with minimal effect on systemic GC biomarkers. In addition, in a mouse model of collagen-induced arthritis, single-dose administration of the ADC, delivered at disease onset, was able to completely inhibit arthritis for greater than 30 days, whereas an anti-TNF monoclonal antibody only partially inhibited disease. ADC treatment at the peak of disease was also able to attenuate the arthritic phenotype. Clinical data for a human anti-TNF-GRM ADC (ABBV-3373) from a single ascending dose phase 1 study in healthy volunteers demonstrated antibody-like pharmacokinetic profiles and a lack of impact on serum cortisol concentrations at predicted therapeutic doses. These data suggest that an anti-TNF-GRM ADC may provide improved efficacy beyond anti-TNF alone in immune mediated diseases while minimizing systemic side effects associated with standard GC treatment.

Structure, recombinant expression and mutagenesis studies of the catalase with oxidase activity from Scytalidium thermophilum.

Scytalidium thermophilum produces a catalase with phenol oxidase activity (CATPO) that catalyses the decomposition of hydrogen peroxide into oxygen and water and also oxidizes various phenolic compounds. A codon-optimized catpo gene was cloned and expressed in Escherichia coli. The crystal structures of native and recombinant S. thermophilum CATPO and two variants, H82N and V123F, were determined at resolutions of 2.7, 1.4, 1.5 and 1.9 Å, respectively. The structure of CATPO reveals a homotetramer with 698 residues per subunit and with strong structural similarity to Penicillium vitale catalase. The haem component is cis-hydroxychlorin γ-spirolactone, which is rotated 180° with respect to small-subunit catalases. The haem-binding pocket contains two highly conserved water molecules on the distal side. The H82N mutation resulted in conversion of the native d-type haem to a b-type haem. Kinetic studies of the H82N and V123F mutants indicate that both activities are likely to be associated with the haem centre and suggest that the secondary oxidase activity may be a general feature of catalases in the absence of hydrogen peroxide.

Investigating the active centre of the Scytalidium thermophilum catalase.

Almost all monofunctional haem catalases contain a highly conserved core containing the active site, which is connected to the exterior of the enzyme by three channels. These channels have been identified as potential routes for substrate flow and product release. To further investigate the role of these molecular channels, a series of mutants of Scytalidium thermophilum catalase were generated. The three-dimensional structures of four catalase variants, N155A, V123A, V123C and V123T, have been determined at resolutions of 2.25, 1.93, 1.9 and 1.7 Å, respectively. The V123C variant contains a new covalent bond between the S atom of Cys123 and the imidazole ring of the essential His82. This variant enzyme has only residual catalase activity and contains haem b instead of the normal haem d. The H82A variant demonstrates low catalase and phenol oxidase activities (0.2 and 20% of those of recombinant wild-type catalase-phenol oxidase, respectively). The N155A and N155H variants exhibit 4.5 and 3% of the wild-type catalase activity and contain haem d, showing that Asn155 is essential for catalysis but is not required for the conversion of haem b to haem d. Structural analysis suggests that the cause of the effect of these mutations on catalysis is the disruption of the ability of dioxygen substrates to efficiently access the active site. Additional mutants have been characterized biochemically to further probe the roles of the different channels. Introducing smaller or polar side chains in place of Val123 reduces the catalase activity. The F160V, F161V and F168V mutants show a marked decrease in catalase activity but have a much lower effect on the phenol oxidase activity, despite containing substoichiometric amounts of haem.

A urea channel from Bacillus cereus reveals a novel hexameric structure.

Urea is exploited as a nitrogen source by bacteria, and its breakdown products, ammonia and bicarbonate, are employed to counteract stomach acidity in pathogens such as Helicobacter pylori. Uptake in the latter is mediated by UreI, a UAC (urea amide channel) family member. In the present paper, we describe the structure and function of UACBc, a homologue from Bacillus cereus. The purified channel was found to be permeable not only to urea, but also to other small amides. CD and IR spectroscopy revealed a structure comprising mainly α-helices, oriented approximately perpendicular to the membrane. Consistent with this finding, site-directed fluorescent labelling indicated the presence of seven TM (transmembrane) helices, with a cytoplasmic C-terminus. In detergent, UACBc exists largely as a hexamer, as demonstrated by both cross-linking and size-exclusion chromatography. A 9 Å (1 Å=0.1 nm) resolution projection map obtained by cryo-electron microscopy of two-dimensional crystals shows that the six protomers are arranged in a planar hexameric ring. Each exhibits six density features attributable to TM helices, surrounding a putative central channel, while an additional helix is peripherally located. Bioinformatic analyses allowed individual TM regions to be tentatively assigned to the density features, with the resultant model enabling identification of residues likely to contribute to channel function.

Optimization of Drug-Linker to Enable Long-term Storage of Antibody-Drug Conjugate for Subcutaneous Dosing.

To facilitate subcutaneous dosing, biotherapeutics need to exhibit properties that enable high-concentration formulation and long-term stability in the formulation buffer. For antibody-drug conjugates (ADCs), the introduction of drug-linkers can lead to increased hydrophobicity and higher levels of aggregation, which are both detrimental to the properties required for subcutaneous dosing. Herein we show how the physicochemical properties of ADCs could be controlled through the drug-linker chemistry in combination with prodrug chemistry of the payload, and how optimization of these combinations could afford ADCs with significantly improved solution stability. Key to achieving this optimization is the use of an accelerated stress test performed in a minimal formulation buffer.

Discovery of ABBV-154, an anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody-Drug Conjugate (iADC).

Stable attachment of drug-linkers to the antibody is a critical requirement, and for maleimide conjugation to cysteine, it is achieved by ring hydrolysis of the succinimide ring. During ADC profiling in our in-house property screening funnel, we discovered that the succinimide ring open form is in equilibrium with the ring closed succinimide. Bromoacetamide (BrAc) was identified as the optimal replacement, as it affords stable attachment of the drug-linker to the antibody while completely removing the undesired ring open-closed equilibrium. Additionally, BrAc also offers multiple benefits over maleimide, especially with respect to homogeneity of the ADC structure. In combination with a short, hydrophilic linker and phosphate prodrug on the payload, this afforded a stable ADC (ABBV-154) with the desired properties to enable long-term stability to facilitate subcutaneous self-administration.

Recombinant production of the therapeutic peptide lunasin.

BACKGROUND: Lunasin is a chemopreventive peptide produced in a number of plant species. It comprises a helical region with homology to a region of chromatin binding proteins, an Arg-Gly-Asp cell adhesion motif and eight aspartic acid residues. In vitro studies indicate that lunasin suppresses chemical and oncogene driven transformation of mammalian cells. We have explored efficient recombinant production of lunasin by exploiting the Clostridium thermocellum CipB cellulose binding domain (CBD) as a fusion partner protein. RESULTS: We used a pET28 vector to express a CBD-lunasin fusion with a hexahistidine tag and Tobacco Etch Virus protease site, to allow protease-mediated release of native lunasin. Autoinduction in E. coli BL21 (DE3) Star cells achieved expression of 3.35 g/L of CBD-lunasin fusion protein. The final yield of lunasin was 210 mg/L corresponding to 32% of the theoretical yield. Purification by cellulose binding and nickel affinity chromatography were tested with the latter proving more satisfactory.The effects of CBD-lunasin expression on growth and morphology of the E. coli cells were examined by light and electron microscopy revealing an altered morphology in a proportion of cells. Cell division appeared to be inhibited in these cells resulting in elongated, non-septated cells. CONCLUSIONS: The use of CBD as a fusion partner gave high protein yields by autoinduction, with lunasin release by TEV protease cleavage. With some optimisation this approach could provide a potentially valuable route for production of this therapeutic peptide. Over-expression in the host cells manifest as a cell division defect in a population of the cells, presumably mimicking some aspect of the chemopreventive function observed in mammalian cells.