Production of human vitronectin in Nicotiana benthamiana using the INPACT hyperexpression platform.

Human vitronectin (hVN) is a glycoprotein that functions as a cell adhesion molecule and a regulator of coagulation in blood plasma and the extracellular matrix. In vitro, hVN is added to serum-free media in order to promote the adhesion of animal cells to tissue culture surfaces and the proliferation of undifferentiated stem cells. Here, we report the production of hVN in Nicotiana benthamiana using the inducible In Plant ACTivation (INPACT) hyperexpression platform. N. benthamiana plants were transformed with an INPACT expression cassette encoding hVN, and both the Tobacco yellow dwarf virus Rep/RepA activator and Tomato bushy stunt virus p19 gene under the transcriptional control of the ethanol-inducible AlcR:alcA gene switch. hVN expression was maximal 4-5 days postactivation of the INPACT platform with a dilute ethanol solution, and crude yields of the recombinant protein reached a maximum of 643 ± 78 mg/kg fresh weight. A three-stage purification protocol was developed using heparin and polyhistidine tag affinity binding and size exclusion filtration, resulting in a plant-made hVN product of >90% purity. Storage conditions for plant-made hVN were identified that maximized the capacity of the recombinant protein to promote cell adhesion. Critically, plant-made hVN was shown to be functionally equivalent to commercial, plasma-derived hVN at promoting one-half maximal attachment of murine fibroblast cells (BALB-C/3T3) in serum-free medium at <0.1 µg/cm2 to tissue culture plasticware. The INPACT platform represents an attractive means of producing large quantities of functional, animal-free hVN for in vitro applications.

Investigation of the variable In(Lu) phenotype caused by KLF1 variants.

INTRODUCTION: KLF1 is an essential transcriptional activator that drives erythropoiesis. KLF1 variants can result in the Inhibitor of Lutheran, or In(Lu), phenotype where red blood cells (RBCs) have reduced BCAM (LU) and CD44 (IN). Other RBC surface molecules also have changed expression; however, there is controversy in the literature regarding which are truly impacted. We aimed to investigate KLF1 variants in the Australian population. STUDY DESIGN AND METHODS: In(Lu) samples were sourced through screening and through the RBC reference laboratory. Blood donor samples (8036) were screened to identify weakened/absent Lub antigen. Samples were genotyped by massively parallel sequencing, while surface carbohydrates and blood group molecules were assessed by flow cytometry. Hemoglobin (Hb) types were analyzed by high-performance liquid chromatography. RESULTS: Four of 8036 donors were identified to be In(Lu), and two previously identified In(Lu) samples were provided from the RBC reference laboratory. Five different KLF1 variants were identified; two were novel: c.954G>C/p.Trp318Cys and c.421C>T/p.Arg141*. BCAM and CD44 were reduced in all samples, consistent with previous reports. As a group, In(Lu) RBCs had reduced CD35 (KN), ICAM4 (LW), and CD147 (OK), and demonstrated increased binding of lectins ECA and SNAI. One In(Lu) sample had elevated HbF and another elevated HbA2. CONCLUSION: Different KLF1 variants may potentially produce variable phenotypes. A framework for investigating KLF1 variants and their phenotypic impact has been provided. In the future, given available international databases, further testing algorithms (as advocated here) will allow for correlation of phenotype with genotype and therefore accurately document this variability between KLF1 variants.

Patient-reported outcomes in patients with overactive bladder treated with mirabegron and tolterodine in a prospective, double-blind, randomized, two-period crossover, multicenter study (PREFER).

BACKGROUND: The PREFER study was an assessment of medication tolerability, treatment preference and symptom improvement during treatment with mirabegron (M) and tolterodine (T) extended release (ER) in patients with overactive bladder (OAB). In this analysis of PREFER, patient-reported outcomes (PROs) were assessed during treatment. METHODS: PREFER was a two-period, 8-week crossover, double-blind, phase IV study (NCT02138747) of treatment-naïve adults with OAB ≥3 months randomized to 1 of 4 treatment sequences (M/T; T/M; M/M; T/T), separated by a 2-week washout. Tolterodine ER was dosed at 4 mg for 8 weeks and mirabegron was dosed at 25 mg for 4 weeks then increased to 50 mg for the next 4 weeks. At each visit, PROs related to treatment satisfaction, quality of life and symptom bother were assessed using the OAB Satisfaction (OAB-S; 3 independent scales/5 single-item overall assessments), OAB-q (total health-related QoL [HRQoL] and subscales [Sleep, Social, Coping, Concern] and Symptom Bother scale) and Patient Perception of Bladder Condition (PPBC) questionnaires. Responder rates were reported for OAB-q subscales based on a minimal important difference (MID; ≥ 10-point improvement) and OAB-S Medication Tolerability score ≥ 90. RESULTS: In total, 358 randomized patients received ≥1 dose of double-blind study medication and completed ≥1 post-baseline value (OAB-S scale, OAB-q, PPBC): M/T (n = 154), T/M (n = 144), M/M (n = 30) or T/T (n = 30). At end of treatment (EoT), mirabegron and tolterodine ER were associated with similar mean improvements in 7 of the 8 OAB-S scores investigated, OAB-q scales and PPBC. A higher percentage of patients achieved clinically relevant improvements (MID) in OAB-q scales and OAB-S Medication Tolerability score during treatment with mirabegron than tolterodine ER. CONCLUSIONS: On average, patients with OAB experienced improvements in treatment satisfaction, HRQoL and symptom bother that were of a similar magnitude during treatment with mirabegron or tolterodine ER. However, during mirabegron treatment, patients were more likely to achieve clinically relevant improvements in tolerability and HRQoL (as measured by the MID for the OAB-q or an OAB-S Medication Tolerability score ≥ 90) than during tolterodine ER treatment. TRIAL REGISTRATION: NCT02138747 ; registered May 13, 2014.

A prospective, double-blind, randomized, two-period crossover, multicenter study to evaluate tolerability and patient preference between mirabegron and tolterodine in patients with overactive bladder (PREFER study).

INTRODUCTION AND HYPOTHESIS: The objective of this study was to assess the tolerability and treatment preference in patients with overactive bladder (OAB) treated with mirabegron or tolterodine. METHODS: This was a two-period, 8-week crossover, double-blind, phase IV study (PREFER; NCT02138747) in treatment-naive adults with OAB for 3 months or longer randomized to one of four treatment sequences in a 5:5:1:1 ratio (mirabegron/tolterodine, tolterodine/mirabegron, mirabegron/mirabegron, or tolterodine/tolterodine), separated by a washout period of 2 weeks. The primary endpoint was drug tolerability using the Medication Tolerability scale of the OAB Treatment Satisfaction (OAB-S) questionnaire at end of treatment (EoT). Period-by-treatment interactions were analyzed to determine any effect of drug order. Patient preference, change from baseline in OAB symptoms, and treatment-emergent adverse events (TEAEs) were assessed. RESULTS: A total of 358 randomized patients completed the OAB-S Medication Tolerability scale questionnaire at one or more visits after the baseline evaluation. The mean (95% CI) OAB-S Medication Tolerability scores were significantly higher (better tolerability) for mirabegron (86.29 [83.50, 89.08]) than for tolterodine (83.40 [80.59, 86.20]; p = 0.004). The period-by-treatment interaction was not significant (p = 0.955). Improvements in OAB-S Medication Tolerability scores at EoT were more evident in women, patients aged ≥65 years, and in patients without baseline incontinence, and were greater with mirabegron than with tolterodine extended release. There were no significant differences in patient preference or improvements in OAB symptoms. Significant differences in favor of mirabegron were observed for anticholinergic TEAEs (20.4% vs. 27.4%; p = 0.042) and specifically for gastrointestinal disorders (14.7% vs. 22.5%; p = 0.015). CONCLUSIONS: Tolerability of mirabegron was significantly higher than that of tolterodine, and patient preference and improvements in OAB symptoms were comparable. Both treatments were well tolerated; however, anticholinergic side effects were higher with tolterodine.

Prediction of a novel internal rearrangement of the insulin receptor.

The insulin receptor (IR) plays critical roles in metabolism and growth, directed by the binding of insulin. Decades of research to understand the mechanism of insulin binding and activation of the IR have identified a region of the receptor, the C-terminal (CT) peptide, to be crucial for insulin binding. In particular, a truncated IR consisting of the first three domains fused to the CT peptide was found to bind insulin with nanomolar affinity, with undetectable binding in the absence of fused or soluble CT peptide. Problematically, all current crystal structures of the IR indicate the fusion point of the CT peptide to the three domains is located far from the position of the CT peptide as resolved in such structures. We have attempted to address this problem using molecular modelling and dynamics simulations. The results led to the identification of a potential inter-domain interaction between the L2 domain and the CT peptide that is not observed in any of the crystal structures of the IR. Investigations into this new interaction found a conformational change that could potentially be in response to insulin binding. Additionally, further simulation work with the new conformation demonstrated its compatibility with the position and orientation of insulin from the latest insulin-bound IR crystal structure.

Identification and Validation of a Salivary Protein Panel to Detect Heart Failure Early.

BACKGROUND: Over 26 million people suffer from heart failure (HF) globally. Current diagnosis of HF relies on clinical evaluation, blood assays and imaging techniques. Our aim is to develop a diagnostic assay to detect HF in at risk individuals within the community using human saliva as a medium, potentially leading to a simple, safe early warning system. METHODS: Saliva samples were collected from healthy controls (n=36) and HF patients (n=75). Salivary proteome profiles were analysed by Sequential Window Acquisition of All Theoretical fragment ion spectra - Mass Spectrometry (SWATH-MS). A total of 738 proteins were quantified and 177 proteins demonstrated significant differences between HF patients and healthy controls. Candidate biomarkers were chosen based on their abundance and difference between the two cohorts. A multi-protein panel was developed using logistic regression analysis. The diagnostic performance of the multi-protein panel was assessed using receiver operative characteristic curves. The candidate proteins were further confirmed, using western blot analysis, and validated technically, using an independent biological cohort. RESULTS: A group of six proteins were chosen in the discovery phase as potential candidates based on their differences in the abundance between the two cohorts. During the validation phase, two of the proteins were not detected with western blotting and as such were removed. The final panel consists of four proteins with sensitivity of 83.3%, specificity of 62.5% with an area under ROC curve of 0.78 in discriminating healthy controls from NYHA class I/II HF patients, and was validated in a second independent cohort study. CONCLUSION: Analysis of salivary proteome using SWATH-MS revealed novel HF-specific protein candidates yielding high diagnostic performance. A multi-centre longitudinal clinical trial will be the next step before clinical implementation of this panel.

Countering cooperative effects in protease inhibitors using constrained beta-strand-mimicking templates in focused combinatorial libraries.

A major problem in de novo design of enzyme inhibitors is the unpredictability of the induced fit, with the shape of both ligand and enzyme changing cooperatively and unpredictably in response to subtle structural changes within a ligand. We have investigated the possibility of dampening the induced fit by using a constrained template as a replacement for adjoining segments of a ligand. The template preorganizes the ligand structure, thereby organizing the local enzyme environment. To test this approach, we used templates consisting of constrained cyclic tripeptides, formed through side chain to main chain linkages, as structural mimics of the protease-bound extended beta-strand conformation of three adjoining amino acid residues at the N- or C-terminal sides of the scissile bond of substrates. The macrocyclic templates were derivatized to a range of 30 structurally diverse molecules via focused combinatorial variation of nonpeptidic appendages incorporating a hydroxyethylamine transition-state isostere. Most compounds in the library were potent inhibitors of the test protease (HIV-1 protease). Comparison of crystal structures for five protease-inhibitor complexes containing an N-terminal macrocycle and three protease-inhibitor complexes containing a C-terminal macrocycle establishes that the macrocycles fix their surrounding enzyme environment, thereby permitting independent variation of acyclic inhibitor components with only local disturbances to the protease. In this way, the location in the protease of various acyclic fragments on either side of the macrocyclic template can be accurately predicted. This type of templating strategy minimizes the problem of induced fit, reducing unpredictable cooperative effects in one inhibitor region caused by changes to adjacent enzyme-inhibitor interactions. This idea might be exploited in template-based approaches to inhibitors of other proteases, where a beta-strand mimetic is also required for recognition, and also other protein-binding ligands where different templates may be more appropriate.

Identification, characterisation and specificity of a cell wall lytic enzyme from Lactobacillus fermentum BR11.

Screening of a genomic library with an antiserum raised against whole Lactobacillus fermentum BR11 cells identified a clone expressing an immunoreactive 37-kDa protein. Analysis of the 3010-bp DNA insert contained within the clone revealed four open reading frames (ORFs). One ORF encodes LysA, a 303 amino acid protein which has up to 35% identity with putative endolysins from prophages Lj928 and Lj965 from Lactobacillus johnsonii and Lp1 and Lp2 from Lactobacillus plantarum as well as with the endolysin of Lactobacillus gasseri bacteriophage Phiadh. The immunoreactive protein was shown to be encoded by a truncated ORF downstream of lysA which has similarity to glutamyl-tRNA synthetases. The N-terminus of LysA has sequence similarity with N-acetylmuramidase catalytic domains while the C-terminus has sequence similarity with putative cell envelope binding bacterial SH3b domains. C-terminal bacterial SH3b domains were identified in the majority of Lactobacillus bacteriophage endolysins. LysA was expressed in Escherichia coli and unusually was found to have a broad bacteriolytic activity range with activity against a number of different Lactobacillus species and against Lactococcus lactis, streptococci and Staphylococcus aureus. It was found that LysA is 2 and 8000 times more active against L. fermentum than L. lactis and Streptococcus pyogenes, respectively.

Cystine uptake prevents production of hydrogen peroxide by Lactobacillus fermentum BR11.

BspA is an abundant surface protein from Lactobacillus fermentum BR11, and is required for normal cystine uptake. In previous studies, a mutant strain deficient in BspA (L. fermentum PNG201) was found to be sensitive to oxidative stress. In this study, the biochemical basis for this was explored. It was found that under aerobic batch culture conditions in de Mann-Rogosa-Sharpe medium, both L. fermentum BR11 and PNG201 entered stationary phase due to hydrogen peroxide accumulation. However, this took place at a lower optical density for PNG201 than for BR11. Measurements of hydrogen peroxide levels revealed that the BspA mutant strain overproduces this compound. Addition of 6 mM cystine to aerobic cultures was found to prevent hydrogen peroxide production by both the BR11 and PNG201 strains, but lower cystine concentrations depressed hydrogen peroxide production in BR11 more efficiently than in PNG201. Each mole of cystine was able to prevent the production of several moles of hydrogen peroxide by L. fermentum BR11, suggesting that hydrogen peroxide breakdown is dependent upon a thiol that cycles between reduced and oxidized states. It was concluded that peroxide breakdown by L. fermentum BR11 is dependent upon exogenous cystine. It is most probable that the imported L-cystine is catabolized by a cystathionine lyase and then converted into a thiol reductant for a peroxidase.

Selective cleavage of human sex hormone-binding globulin by kallikrein-related peptidases and effects on androgen action in LNCaP prostate cancer cells.

Stimulation of the androgen receptor via bioavailable androgens, including testosterone and testosterone metabolites, is a key driver of prostate development and the early stages of prostate cancer. Androgens are hydrophobic and as such require carrier proteins, including sex hormone-binding globulin (SHBG), to enable efficient distribution from sites of biosynthesis to target tissues. The similarly hydrophobic corticosteroids also require a carrier protein whose affinity for steroid is modulated by proteolysis. However, proteolytic mechanisms regulating the SHBG/androgen complex have not been reported. Here, we show that the cancer-associated serine proteases, kallikrein-related peptidase (KLK)4 and KLK14, bind strongly to SHBG in glutathione S-transferase interaction analyses. Further, we demonstrate that active KLK4 and KLK14 cleave human SHBG at unique sites and in an androgen-dependent manner. KLK4 separated androgen-free SHBG into its two laminin G-like (LG) domains that were subsequently proteolytically stable even after prolonged digestion, whereas a catalytically equivalent amount of KLK14 reduced SHBG to small peptide fragments over the same period. Conversely, proteolysis of 5α-dihydrotestosterone (DHT)-bound SHBG was similar for both KLKs and left the steroid binding LG4 domain intact. Characterization of this proteolysis fragment by [(3)H]-labeled DHT binding assays revealed that it retained identical affinity for androgen compared with full-length SHBG (dissociation constant = 1.92 nM). Consistent with this, both full-length SHBG and SHBG-LG4 significantly increased DHT-mediated transcriptional activity of the androgen receptor compared with DHT delivered without carrier protein. Collectively, these data provide the first evidence that SHBG is a target for proteolysis and demonstrate that a stable fragment derived from proteolysis of steroid-bound SHBG retains binding function in vitro.

Proteomics in chronic wound research: potentials in healing and health.

Chronic wounds, such as venous and diabetic leg ulcers, represent a significant health and financial burden to individuals and healthcare systems. In worst-case scenarios this condition may require the amputation of an affected limb, with significant impact on patient quality of life and health. Presently, there are no clinical biochemical analyses used in the diagnosis and management of this condition; moreover few biochemical therapies are accessible to patients. This presents a significant challenge in the efficient and efficacious treatment of chronic wounds by medical practitioners. A number of protein-centric investigations have analyzed the wound environment and implicated a suite of molecular species predicted to be involved in the initiation or perpetuation of the condition. However, comprehensive proteomic investigation is yet to be engaged in the analysis of chronic wounds for the identification of molecular diagnostic/prognostic markers of healing or therapeutic targets. This review examines clinical chronic wound research and recommends a path toward proteomic investigation for the discovery of medically significant targets. Additionally, the Supporting Information documents associated with this review provide the first comprehensive summary of protein-centric, small molecule and elemental analyses in clinical chronic wound research.

Substrate-guided design of a potent and selective kallikrein-related peptidase inhibitor for kallikrein 4.

Human kallikrein-related peptidase 4 (KLK4/prostase), a trypsin-like serine protease, is a potential target for prostate cancer treatment because of its proteolytic ability to activate many tumorigenic and metastatic pathways including the protease activated receptors (PARs). Currently there are no KLK4-specific small-molecule inhibitors available for therapeutic development. Here we re-engineer the naturally occurring sunflower trypsin inhibitor to selectively block the proteolytic activity of KLK4 and prevent stimulation of PAR activity in a cell-based system. The re-engineered inhibitor was designed using a combination of molecular modeling and sparse matrix substrate screening.

Crystal structures of highly constrained substrate and hydrolysis products bound to HIV-1 protease. Implications for the catalytic mechanism.

HIV-1 protease is a key target in treating HIV infection and AIDS, with 10 inhibitors used clinically. Here we used an unusual hexapeptide substrate, containing two macrocyclic tripeptides constrained to mimic a beta strand conformation, linked by a scissile peptide bond, to probe the structural mechanism of proteolysis. The substrate has been cocrystallized with catalytically active synthetic HIV-1 protease and an inactive isosteric (D25N) mutant, and three-dimensional structures were determined (1.60 A). The structure of the inactive HIVPR(D25N)/substrate complex shows an intact substrate molecule in a single orientation that perfectly mimics the binding of conventional peptide ligands of HIVPR. The structure of the active HIVPR/product complex shows two monocyclic hydrolysis products trapped in the active site, revealing two molecules of the N-terminal monocyclic product bound adjacent to one another, one molecule occupying the nonprime site, as expected, and the other monocycle binding in the prime site in the reverse orientation. The results suggest that both hydrolysis products are released from the active site upon cleavage and then rebind to the enzyme. These structures reveal that N-terminal binding of ligands is preferred, that the C-terminal site is more flexible, and that HIVPR can recognize substrate shape rather than just sequence alone. The product complex reveals three carboxylic acids in an almost planar orientation, indicating an unusual hexagonal homodromic complex between three carboxylic acids. The data presented herein regarding orientation of catalytic aspartates support the cleavage mechanism proposed by Northrop. The results imply strategies for design of inhibitors targeting the N-terminal side of the cleavage site or taking advantage of the flexibility in the protease domain that accommodates substrate/inhibitor segments C-terminal to the cleavage site.

Seminal fluid characterization for male fertility and prostate cancer: kallikrein-related serine proteases and whole proteome approaches.

The kallikrein-related ( KLK) protease gene family encodes a subgroup of 15 serine proteases that includes prostate-specific antigen (PSA) or KLK3, the well-known biomarker for prostate cancer. PSA is also a major component of seminal fluid. To date, 10 other KLK serine proteases have been documented as present in seminal fluid (KLKs 1, 2, 4, 5, 6, 8, 10, 11, 13, and 14) and, like PSA, have the potential to contribute to male fertility, either directly or indirectly, by means of their proteolytic activity on seminal coagulum proteins. These KLK enzymes arise predominantly from the glandular epithelium of the prostate and are secreted into the lumen of the prostatic ducts that empty into the urethra upon ejaculation. Given their prostatic origin, they are also being considered increasingly as diagnostic/prognostic targets for prostate cancer. This article reviews the literature on seminal fluid PSA and more recent reports on the detection of other KLKs enzymes in this milieu, and their potential roles in male fertility and prostate cancer. We also discuss recent efforts to determine the proteomic profile of seminal fluid to identify new biomarkers for prostate disease.

BspA (CyuC) in Lactobacillus fermentum BR11 is a highly expressed high-affinity L-cystine-binding protein.

The BspA protein of Lactobacillus fermentum BR11 (BR11) is a cell envelope constituent that is similar to known solute-binding proteins and putative adhesins. BspA is required for L-cystine uptake and oxidative defense and is likely to be an L-cystine-binding protein. The aim of this study was to directly measure L-cystine-BspA binding and BspA expression. De-energized BR11 cells bound radiolabelled L-cystine with a Kd of 0.2 microM. A bspA mutant could not bind L-cystine. L-cystine-BR11 binding was unaffected by large excesses of L-glutamine, L-methionine, or collagen, indicating L-cystine specificity. BR11 and the bspA mutant were identical in their abilities to bind L-cysteine, indicating that L-cysteine is not a BspA ligand. BspA expression levels were deduced from radiolabelled L-cystine binding and it was found that there are 1-2 x 10(5) BspA molecules per cell, and that expression is slightly higher under oxidizing conditions. It is proposed that BspA be renamed CyuC.

Peptide surface display and secretion using two LPXTG-containing surface proteins from Lactobacillus fermentum BR11.

A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from Lactobacillus fermentum BR11 has been identified by using an antiserum raised against whole L. fermentum BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from Streptococcus agalactiae, while the other protein, Mlp, is similar to the mucus binding protein Mub from Lactobacillus reuteri. It was shown that multiple copies of mlp exist in the genome of L. fermentum BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in L. fermentum. The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His(6)). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The mlp promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His(6) and CFTR peptides or His(6) peptide, respectively. Therefore, these newly described proteins from L. fermentum BR11 have potential as protein production and targeting vectors.

Identification and characterization of the novel LysM domain-containing surface protein Sep from Lactobacillus fermentum BR11 and its use as a peptide fusion partner in Lactobacillus and Lactococcus.

Examination of supernatant fractions from broth cultures of Lactobacillus fermentum BR11 revealed the presence of a number of proteins, including a 27-kDa protein termed Sep. The amino-terminal sequence of Sep was determined, and the gene encoding it was cloned and sequenced. Sep is a 205-amino-acid protein and contains a 30-amino-acid secretion signal and has overall homology (between 39 and 92% identity) with similarly sized proteins of Lactobacillus reuteri, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus agalactiae, and Lactobacillus plantarum. The carboxy-terminal 81 amino acids of Sep also have strong homology (86% identity) to the carboxy termini of the aggregation-promoting factor (APF) surface proteins of Lactobacillus gasseri and Lactobacillus johnsonii. The mature amino terminus of Sep contains a putative peptidoglycan-binding LysM domain, thereby making it distinct from APF proteins. We have identified a common motif within LysM domains that is shared with carbohydrate binding YG motifs which are found in streptococcal glucan-binding proteins and glucosyltransferases. Sep was investigated as a heterologous peptide expression vector in L. fermentum, Lactobacillus rhamnosus GG and Lactococcus lactis MG1363. Modified Sep containing an amino-terminal six-histidine epitope was found associated with the cells but was largely present in the supernatant in the L. fermentum, L. rhamnosus, and L. lactis hosts. Sep as well as the previously described surface protein BspA were used to express and secrete in L. fermentum or L. rhamnosus a fragment of human E-cadherin, which contains the receptor region for Listeria monocytogenes. This study demonstrates that Sep has potential for heterologous protein expression and export in lactic acid bacteria.