Recombinant Polypeptide Research Articles

Bioinspired polypeptide as building blocks for multifunctional material design

The exceptional mechanical properties of spider silks from their extraordinary natural design make them outstanding biomaterial candidates. Herein, inspired by the outer egg sac silk from Nephila pilipes, we introduce a gradient of recombinant biomimetic polypeptides (BMPPs) through a semi-rational design. The synthesized BMPPs show good water solubility with dynamic conformations of random coil/helix. Both biomimetic nanofiber membranes and macrofiber yarns are fabricated using BMPPs as blend with polycaprolactone (PCL) through electrospinning. The resulting nanofiber membrane yields almost double tensile strength (22.7 ± 3.3 MPa) compared to that of PCL (12.5 ± 1.4 MPa), without sacrifice of toughness. The as-fabricated microfiber yarns could accommodate both improvement of tensile strength (208 ± 8.3 MPa, almost twice of PCL value) and toughness (163 ±13 MJ/m3, more than twice of PCL value), suggesting the crosslink role of BMPP in PCL network. By fusion of cell-binding domains in the BMPP, the materials also illustrate great potentials into new biomedical materials.

Design of Thermoresponsive Elastin-Like Glycopolypeptides for Selective Lectin Binding and Sorting.

Selective lectin binding and sorting was achieved using thermosensitive glycoconjugates derived from recombinant elastin-like polypeptides (ELPs) in simple centrifugation-precipitation assays. A recombinant ELP, (VPGXG)40, containing periodically spaced methionine residues was used to enable chemoselective postsynthetic modification via thioether alkylation using alkyne functional epoxide derivatives. The resulting sulfonium groups were selectively demethylated to give alkyne functionalized homocysteine residues, which were then reacted with azido-functionalized monosaccharides to obtain ELP glycoconjugates with periodic saccharide functionality. These modifications were also found to allow modulation of ELP temperature dependent water solubility. The multivalent ELP glycoconjugates were evaluated for specific recognition, binding and separation of the lectin Ricinus communis agglutinin (RCA120) from a complex protein mixture. RCA120 and ELP glycoconjugate interactions were evaluated using laser scanning confocal microscopy and dynamic light scattering. Due to the thermoresponsive nature of the ELP glycoconjugates, it was found that heating a mixture of galactose-functionalized ELP and RCA120 in complex media selectively yielded a phase separated pellet of ELP-RCA120 complexes. Based on these results, ELP glycoconjugates show promise as designer biopolymers for selective protein binding and sorting.

Streptococcus pneumoniae coinfection dictates susceptibility to oral Listeria monocytogenes through suppressive Natural Killer (NK) cell activity

Abstract Coinfection with viral and bacterial pathogens is a major health concern and results in significant morbidity and mortality. Recent research has aimed to understand the complex interplay between multiple pathogens and the immune response during viral:bacterial coinfections. However, studies using bacterial-bacterial coinfections are lacking. Streptococcus pneumoniae (Spn) and Listeria monocytogenes (Lm) are both bacterial pathogens that are capable of causing severe infections in young, elderly, and immunocompromised populations. Here we show that coinfection with intratracheal (IT) Spn increases susceptibility of typically resistant mice to oral Lm infection. On day 3 post infection (p.i.), the presence of Spn increased oral Lm burdens in the spleen, liver, and lungs of coinfected C57/BL6 mice. In addition, while the anti-inflammatory cytokine Interleukin-10 (IL-10) is limited during oral Lm infection, Spn infection induced the production of IL-10 from Natural Killer (NK) cells, and that this correlated with an increase in Lm burdens during coinfection. To assess the importance of NK cell IL-10 in the absence of an ongoing infection, mice were inoculated with a recombinant Lm polypeptide, designated L1S, which stimulates NK cell IL-10 production. In the presence of L1S, the induction of NK cell IL-10 similarly increased systemic burdens of oral Lm. Lastly, BMMs stimulated with rIL-10 and infected with Lm have increased bacterial burdens when compared to untreated but infected controls. Overall, our data show that coinfection impairs host protection through the induction of NK cell production of IL-10 and dictates susceptibility to oral Lm infection.

Human Granulocyte-Macrophage Colony-Stimulating Factor Fused to Elastin-Like Polypeptides Assembles Biologically-Active Nanoparticles.

Human granulocyte-macrophage colony-stimulating factor (hGMCSF) is crucial in the immune system as it stimulates survival, proliferation, differentiation, and functional activation of myeloid hematopoietic cells. hGMCSF is integral to approved therapies, including monoclonal antibodies against checkpoint inhibitors, chimeric antigen receptors, and prevention of chemotherapy-induced neutropenia. Recombinant hGMCSF can be purified from Escherichia. coli; however, it forms inclusion bodies that require solubilization and refolding. Alternatively, this manuscript describes its fusion with an elastin-like polypeptide (ELP). Previously reported as purification tags and solubility enhancers, ELPs are recombinant polypeptides that undergo reversible temperature-dependent phase separation. This report is the first to show that fusion to an ELP enables direct purification of hGMCSF fusions from the soluble fraction of bacterial lysate. Surprisingly, these ELP-fusions assemble stable, small, spherical nanoparticles that maintain pro-mitotic activity of hGMCSF. These nanoparticles exhibit ELP-mediated phase separation; however, nanoparticle assembly significantly increases the entropic and enthalpic cost of phase separation compared to ELP alone. The attachment of a high molecular weight ELP to a difficult-to-express protein, like hGMCSF, appears to be a useful strategy to stabilize bioactive, protein-based nanoparticles, which may have broad applications in medicine and biology.

Significance of a family-6 carbohydrate-binding module in a modular feruloyl esterase for removing ferulic acid from insoluble wheat arabinoxylan

Ruminiclostridium josui Fae1A is a modular enzyme consisting of an N-terminal signal peptide, family-1 carbohydrate esterase module (CE1), family-6 carbohydrate-binding module (CBM6), and dockerin module in that order. Recombinant CE1 and CBM6 polypeptides were collectively and separately produced as RjFae1A, RjCE1, and RjCBM6. RjFae1A showed higher feruloyl esterase activity than RjCE1 towards insoluble wheat arabinoxylan, but the latter was more active towards small synthetic substrates than the former. This suggests that CBM6 in RjFae1A plays an important role in releasing ferulic acid from the native substrate. RjCBM6 showed a higher affinity for soluble wheat arabinoxylan than for rye arabinoxylan and beechwood xylan in native affinity polyacrylamide gel electrophoresis. Isothermal titration calorimetry analysis demonstrated that RjCBM6 recognized a xylopyranosyl residue at the nonreducing ends of xylooligosaccharides. Moreover, it showed exceptional affinity for 23-α-l-arabinofuranosyl-xylotriose (A2XX) among the tested branched arabinoxylooligosaccharides. Fluorometric titration analysis demonstrated that xylobiose and A2XX competitively bound to RjCBM6, and both bound to the same site in RjCBM6. RjCBM6’s preference for the xylopyranosyl residue at the nonreducing end of xylan chains explains why the positive effect of CBM6 on RjFae1A activity was observed only during short incubation but not after extended incubation.

Sequence-structure characterization of recombinant polypeptides derived from silk fibroin heavy chain.

The molecular conformation of a biomedical material plays a major role in the stability, bioactivity and controlled release of drugs. In order to identify the impact of fragments derived from Bombyx mori silk fibroin on their structures and to develop a new strategy for controlling drug release, we designed several hydrophobic-hydrophilic recombinants (GS16F1, GS16F4, and GS16F8), and investigated their molecular conformations and conformational changes induced by different storage temperatures and pH values. The results showed that the α-helix characteristic peaks were prominent in the fresh freeze-dried powder with increasing F1 repeats. During storage at 4°C, 37°C or 60°C, the β-turns (especially in GS16F8) and α-helixes turned into β-sheets. The β-sheet content in the polypeptides increased with increasing temperature and F1 repeats. Following induction by different pH values, their molecular conformations changed significantly, but not the same as that of powder storage. The content of β-sheets was GS16F1>GS16F4>GS16F8 near the isoelectric point of each polypeptide. With increasing pH value, the β-sheet content of GS16F1 decreased more slowly compared with GS16F4 and GS16F8. These results were satisfactory for structural regulation in the field of drug controlled release research.

Genetically Encoded Stealth Nanoparticles of a Zwitterionic Polypeptide-Paclitaxel Conjugate Have a Wider Therapeutic Window than Abraxane in Multiple Tumor Models.

Small-molecule therapeutics demonstrate suboptimal pharmacokinetics and bioavailability due to their hydrophobicity and size. One way to overcome these limitations-and improve their efficacy-is to use "stealth" macromolecular carriers that evade uptake by the reticuloendothelial system. Although unstructured polypeptides are of increasing interest as macromolecular drug carriers, current recombinant polypeptides in the clinical pipeline typically lack stealth properties. We address this challenge by developing new unstructured polypeptides, called zwitterionic polypeptides (ZIPPs), that exhibit "stealth" behavior in vivo. We show that conjugating paclitaxel to a ZIPP imparts amphiphilicity to the polypeptide chain that is sufficient to drive its self-assembly into micelles. This in turn increases the half-life of paclitaxel by 17-fold compared to free paclitaxel, and by 1.6-fold compared to the nonstealth control, i.e., ELP-paclitaxel. Treatment of mice bearing highly aggressive prostate or colon cancer with a single dose of ZIPP-paclitaxel nanoparticles leads to near-complete eradication of the tumor, and these nanoparticles have a wider therapeutic window than Abraxane, an FDA-approved taxane nanoformulation.

Recombinant Production of Opiorphin Pentapeptide as Tandem Multimers Through Rational Design of Primers

Opiorphin secreted into the human saliva is an endogenous regulator pentapeptide (QRFSR) showing a pain suppressant effect similar to morphine. We report here a strategy of producing tandemly repeated multimers of opiorphin peptide efficiently by expressing in Esherichia coli cells. For improving the expression level and increasing the yield of pentapeptide, 32-mer tandem sequence of opiorphin was generated by using partially complementary primers inserted into pGEX-2T expression vector, following fused with glutathione transferase (GST) gene. Recombinant tandem multimer was easily captured by GST column with a yield of 9.2 mg/L, and then 32-mer opiorphin polypeptide of 28.49 kDa size was cleaved into monomers by CNBr and endoproteinase Asp-N. Opiorphin polypeptides having a molecular weight of about 693 Da and containing 5 amino acids in length were then generated. The amount of cleaved and purified recombinant opiorphin monomers was calculated as 6.2 mg/L by the Bradford assay. Protein expression and cleavage of recombinant polypeptide were confirmed by SDS-PAGE and TLC. Results from this study clearly open a door to the mass production of the opiorphin peptide to use for laboratory research and industrial purposes. The overall gene construction and the direct cloning to an expression vector strategy using partially complementary primers with cleavable linker peptides could be applicable to many small peptides and their analogs for pharmaceutical and clinical application.

Prolonging and enhancing the protective efficacy of the EtMIC3-C-MAR against eimeria tenella through delivered by attenuated salmonella typhimurium

The microneme adhesive repeats (MAR) of Eimeria tenella microneme protein 3 (EtMIC3) are associated with binding to and invasion of host cells. Adhesion and invasion-related proteins or domains are often strongly immunogenic, immune responses mounted against these factors that play a key role in blocking invasion. In the present study, an oral live vaccine consisting of attenuated Salmonella typhimurium X4550 carrying two MAR domains fragment (St-X4550-MAR) was constructed and its protective efficacies were evaluated. The results showed that St-X4550-MAR was more immunogenic and conferred a higher degree of protection than recombinant MAR polypeptide as reflected by increased body weight, decreased oocyst shedding and lesion scores, increased serum IgG and cecal sIgA antibody production, and increasing levels of interferon-γ and interleukin-10. Thus, MAR domains are highly immunogenic and St-X4550-MAR had moderate activity against E. tenella infection by stimulating humoral, mucosal and cellular immunity. Chickens immunized with our constructed live vaccine provided considerable protections as early as at 10 d post-immunization (ACI: 155.17), and maintained higher protection levels at 20 d post-immunization (ACI: 173.66), and at 30 d post-immunization (ACI: 162.4). While the protective efficacy of chickens immunized with the recombinant MAR peptides showed a decreased trend as the post immunization time prolonging. Thus, using live-attenuated S. typhimurium X4550 as a vaccine expression and delivery system can significantly improve the protective efficacy and duration of protective immunity of MAR of EtMIC3.

Expanded toolbox for directing the biosynthesis of macrocyclic peptides in bacterial cells.

The macrocyclization of recombinant polypeptides by means of genetically encodable non-canonical amino acids has recently provided an attractive strategy for the screening and discovery of macrocyclic peptide inhibitors of protein-protein interactions. Here, we report the development of an expanded suite of electrophilic unnatural amino acids (eUAAs) useful for directing the biosynthesis of genetically encoded thioether-bridged macrocyclic peptides in bacterial cells (E. coli). These reagents are shown to provide efficient access to a broad range of macrocyclic peptide scaffolds spanning from 2 to 20 amino acid residues, with the different eUAAs offering complementary reactivity profiles toward mediating short- vs. long-range macrocyclizations. Swapping of the eUAA cyclization module in a cyclopeptide inhibitor of streptavidin and Keap1 led to compounds with markedly distinct binding affinity toward the respective target proteins, highlighting the effectiveness of this strategy toward tuning the structural and functional properties of bioactive macrocyclic peptides. The peptide cyclization strategies reported here expand opportunities for the combinatorial biosynthesis of natural product-like peptide macrocycles in bacterial cells or in combination with display platforms toward the discovery of selective agents capable of targeting proteins and protein-mediated interactions.

A tropomyosin-like Meretrix meretrix Linnaeus polypeptide inhibits the proliferation and metastasis of glioma cells via microtubule polymerization and FAK/Akt/MMPs signaling

Glioblastoma (GBM) represents the most common, aggressive and deadliest primary tumors with poor prognosis as available therapeutic approaches fail to control its aberrant proliferation and high invasiveness. Thus, the therapeutic agents targeting these two characteristics will be more effective. In present study, a novel polypeptide (MM15), which was originally purified from Meretrix meretrix Linnaeus and has been proven to possess potent antitumor activity by our laboratory, was recombinant expressed and identified as a tropomyosin homologous protein. The recombinant polypeptide (re-MM15) could induce the U87 cell cycle arrest in G2/M phase and cell apoptosis by inducing tubulin polymerization. Additionally, re-MM15 displayed the significant inhibition to the migration and invasion of U87 cells through downregulating FAK/Akt/MMPs signaling. Furthermore, the in vivo analysis suggested that re-MM15 significantly blocked tumor growth in U87 xenograft model. Collectively, our results indicated that re-MM15, with anti-GBM properties in vitro and in vivo, has promising potential as a new anticancer candidate for GBM.

Recombinant polypeptide of Mycobacterium leprae as a potential tool for serological detection of leprosy

Current prevention methods for the transmission of Mycobacterium leprae, the causative agent of leprosy, are inadequate as suggested by the rate of new leprosy cases reported. Simple large-scale detection methods for M. leprae infection are crucial for early detection of leprosy and disease control. The present study investigates the production and seroreactivity of a recombinant polypeptide composed of various M. leprae protein epitopes. The structural and physicochemical parameters of this construction were assessed using in silico tools. Parameters like subcellular localization, presence of signal peptide, primary, secondary, and tertiary structures, and 3D model were ascertained using several bioinformatics tools. The resultant purified recombinant polypeptide, designated rMLP15, is composed of 15 peptides from six selected M. leprae proteins (ML1358, ML2055, ML0885, ML1811, ML1812, and ML1214) that induce T cell reactivity in leprosy patients from different hyperendemic regions. Using rMLP15 as the antigen, sera from 24 positive patients and 14 healthy controls were evaluated for reactivity via ELISA. ELISA-rMLP15 was able to diagnose 79.17% of leprosy patients with a specificity of 92.86%. rMLP15 was also able to detect the multibacillary and paucibacillary patients in the same proportions, a desirable addition in the leprosy diagnosis. These results summarily indicate the utility of the recombinant protein rMLP15 in the diagnosis of leprosy and the future development of a viable screening test.

Anti-Nociceptive and Anti-Inflammation Effect Mechanisms of Mutants of Syb-prII, a Recombinant Neurotoxic Polypeptide.

Syb-prII, a recombinant neurotoxic polypeptide, has analgesic effects with medicinal value. Previous experiments indicated that Syb-prII displayed strong analgesic activities. Therefore, a series of in vivo and vitro experiments were designed to investigate the analgesic and anti-inflammatory properties and possible mechanisms of Syb-prII. The results showed that administered Syb-prII-1 and Syb-prII-2 (0.5, 1, 2.0 mg/kg, i.v.) to mice significantly reduced the time of licking, biting, or flicking of paws in two phases in formalin-induced inflammatory nociception. Syb-prII-1 inhibited xylene-induced auricular swelling in a dose-dependent manner. The inhibitory effect of 2.0 mg/kg Syb-prII-1 on the ear swelling model was comparable to that of 200 mg/kg aspirin. In addition, the ELISA and Western blot analysis suggested that Syb-prII-1 and Syb-prII-2 may exert an analgesic effect by inhibiting the expression of Nav1.8 and the phosphorylation of ERK, JNK, and P38. Syb-prII-1 markedly suppressed the expression of IL-1β, IL-6, and TNF-α of mice in formalin-induced inflammatory nociception. We used the patch-clamp technique and investigated the effect of Syb-prII-1 on TTX-resistant sodium channel currents in acutely isolated rat DRG neurons. The results showed that Syb-prII-1 can significantly down regulate TTX-resistant sodium channel currents. In conclusion, Syb-prII mutants may alleviate inflammatory pain by significantly inhibiting the expression of Nav1.8, mediated by the phosphorylation of MAPKs and significant inhibition of TTX-resistant sodium channel currents.

Cloning and expression of the recombinant polypeptide based on the Anaplasma marginale MSP1b protein in Escherichia coli

Cloning and expression of the recombinant polypeptide based on the Anaplasma marginale MSP1b protein in Escherichia coli

Heuristics for the Optimal Presentation of Bioactive Peptides on Polypeptide Micelles.

Bioactive peptides describe a very large group of compounds with diverse functions and wide applications, and their multivalent display by nanoparticles can maximize their activities. However, the lack of a universal nanoparticle platform and design rules for their optimal presentation limits the development and application of peptide ligand-decorated nanoparticles. To address this need, we developed a multivalent nanoparticle platform to study the impact of nanoparticle surface hydrophilicity and charge on peptide targeting and internalization by tumor cells. This system consists of micelles of a recombinant elastin-like polypeptide diblock copolymer (ELPBC) that present genetically encoded peptides at the micelle surface without perturbing the size, shape, stability, or peptide valency of the micelle, regardless of the peptide type. We created the largest extant set of 98 combinations of 15 tumor-homing peptides that are presented on the corona of this ELPBC micelle via 8 different peptide linkers that vary in their length and charge and also created control micelles that present the linker only. Analysis of the structure-function relationship of tumor cell targeting by this set of peptide-decorated nanoparticles enabled us to derive heuristics to optimize the delivery of peptides based on their physicochemical properties and to identify a peptide that is likely to be a widely useful ligand for targeting across nanoparticle types. This study shows that ELPBC micelles are a robust and convenient system for the presentation of diverse peptides and provides useful insights into the appropriate presentation of structurally diverse peptide ligands on nanoparticles based on their physicochemical properties.

Early detection and differential serodiagnosis of Mycoplasma hyorhinis and Mycoplasma hyosynoviae infections under experimental conditions.

Mycoplasma hyorhinis (MHR) and Mycoplasma hyosynoviae (MHS) are common opportunistic pathogens in the upper respiratory tract and tonsils of swine. The identification of the specific species involved in clinical cases using conventional diagnostic methods is challenging. Therefore, a recombinant chimeric polypeptide based on the seven known variable lipoproteins (A-G) specific of MHR and a cocktail of surface proteins detergent-extracted from MHS cultures were generated and their suitability as antemortem biomarkers for serodiagnosis of MHR- and MHS-infection were evaluated by ELISA. M. hyorhinis and MHS ELISA performance, evaluated using serum samples collected over a 56-day observation period from pigs inoculated with MHR, MHS, M. hyopneumoniae, M. flocculare, or Friis medium, varied by assay, targeted antibody isotype, and cutoffs. The progressions of MHR and MHS clinical diseases were evaluated in relation to the kinetics of the isotype-specific antibody response in serum and bacterial shedding in oral fluids during the observation period. In pigs inoculated with MHR, bacterial DNA was detected in one or more of the 5 pens at all sampling points throughout the study, IgA was first detected at DPI 7, one week before the first clinical signs, with both IgA and IgG detected in all samples collected after DPI 14. The peak of MHS shedding (DPI 8) coincided with the onset of the clinical signs, with both IgA and IgG detected in all serum samples collected ≥ DPI 14. This study demonstrated, under experimental conditions, that both ELISAs were suitable for early detection of specific antibodies against MHR or MHS. The diagnostic performance of the MHR and MHS ELISAs varied depending on the selected cutoff and the antibody isotype evaluated. The high diagnostic and analytical specificity of the ELISAs was particularly remarkable. This study also provides insights into the infection dynamics of MHR-associated disease and MHS-associated arthritis not previously described.

Generation of a lentiviral vector system to efficiently express bioactive recombinant human prolactin hormones

The contribution of the pleiotropic hormone Prolactin (PRL) to several physiological and pathological processes is still unknown. To clarify the role of PRL in these processes during the last decade, different human PRL antagonists have been produced to either partially or fully block the wild type hormone activity. In this work, we have cloned these wild type and antagonist sequences in lentivectors (LV) to express them as recombinant self-processing polypeptides by employing a P2A sequence (hPRL-P2A-GFP). We show that these LVs can efficiently transduce and express the hPRL proteins in different cell types and that the P2A sequence does not affect their activities. Additionally, we have tested their activities in paracrine and autocrine cell culture experiments. Our results demonstrate that these recombinant hPRL-P2A proteins are bioactive in both paracrine and autocrine modes, highlighting the potential usefulness of these hPRL-containing LVs for determining the contribution of hPRL to different biological processes.

Functional significance of the conserved C-Terminal VFVNFA motif in the retina-specific ABC transporter, ABCA4, and its role in inherited visual disease

The human retina-specific ATP binding cassette transporter, ABCA4, plays a significant role in the visual cycle. Mutations in the ABCA4 gene result in a broad spectrum of severe, blinding, retinal degenerative diseases, including Stargardt macular dystrophy, fundus flavimaculatus, autosomal recessive (ar)-retinitis pigmentosa, and ar-cone-rod dystrophy. Genetic testing frequently yields novel variants of unknown significance, making accurate prognosis and therapeutic approaches difficult.Recently, we have reported a novel variant of ABCA4 corresponding to a four-nucleotide deletion which led to a premature stop codon and loss of the last 161 amino acids, including the highly-conserved VFVNFA motif. Despite the presence of this motif among other ABCA proteins, knowledge of the functional significance of this sequence remains limited. In this study, we have conducted structural and functional analyses of recombinant ABCA4 polypeptides with altered VFVNFA motifs to evaluate the importance of this sequence. Further investigation of ABCA4 subdomain interactions, using Fluorescence Resonance Energy Transfer, demonstrated a loss of interaction between nucleotide binding domains in the absence of the VFVNFA motif.

Injectable Polypeptide Hydrogel Depot System for Assessment of the Immune Response-Inducing Efficacy of Sustained Antigen Release Alone.

Vaccines typically contain an antigen, delivery system (vehicle), and adjuvant, all of which contribute to inducing a potent immune response. Consequently, design of new vaccines is difficult, because the contributions and interactions of these components are difficult to distinguish. Here, it is aimed to develop an easy-to-use, non-immunogenic, injectable depot system for sustained antigen release that will be suitable for assessing the efficacy of prolonged antigen exposure per se for inducing an immune response. This should mimic real-life infections. Recombinant elastin-like polypeptides with periodic cysteine residues (cELPs) are selected, which reportedly show little or no immunogenicity, as carriers and tetanus toxoid (Ttd) as an antigen. After subcutaneous injection of the mixture, cELP rapidly forms a disulfide cross-linked hydrogel in situ, within which Ttd is physically incorporated, affording a biodegradable antigen depot. A series of Ttd-containing hydrogels is examined. A single injection induces high levels of tetanus antibody with high avidity for at least 20 weeks in mice. The chain length of cELP proves critical, whereas differences in hydrophobicity has little effect, although hydrophilic cELPs are more rapidly biodegraded. This system's ability to distinguish the contribution of sustained antigen release to antibody induction should be helpful for rational design of next-generation vaccines.

PerioVax3, a key antigenic determinant with immunoprotective potential against periodontal pathogen

Treponema (T.) denticola is one of the key etiological agents in the development of periodontitis. The major outer sheath protein (Msp) of T. denticola has been shown to mediate pathogenesis and to facilitate adhesion of T. denticola to mucosal surfaces. This study aimed to find short polypeptides in the amino acid sequence of Msp which may be immunogenic and might elicit protective antisera against T. denticola. The complete msp sequence was divided into six fragments and the corresponding genes were cloned and expressed. Antisera against the polypeptides were raised in rabbits and fragment 3 (F3), hereinafter called PerioVax3 was the most potent fragment of the Msp in terms of yielding high titer antiserum. An adhesion assay was done to examine the inhibitory effects of antisera on the attachment of T. denticola to human gingival fibroblasts (HGFs) and human fibronectin. Antiserum against PerioVax3 significantly inhibited attachment of T. denticola to the substratum. Also, antiserum against PerioVax3 inhibited detachment of HGFs upon T. denticola exposure. To begin examining the clinical relevance of this work, blood samples from 12 sever periodontitis patients were collected and the sera were used in western blotting against the recombinant polypeptides. Periodontitis patient antisera exclusively detected PerioVax3 in western blotting. The data suggest that PerioVax3 carries epitopes that may trigger humoral immunity against T. denticola, which may protect against its adhesion functions. The complexity of periodontitis suggests that PerioVax3 may be considered for testing as a component of an experimental multivalent periodontal vaccine in further preclinical and clinical studies.