A cleavable self‐assembling tag strategy for preparing proteins and peptides with an authentic N‐terminus

Recombinant protein expression and purification remains a central need for biotechnology. Herein, the authors report a streamlined protein and peptide purification strategy using short self‐assembling peptides and a C‐terminal cleavage intein. In this strategy, the fusion protein is first expressed as an aggregate induced by the self‐assembling peptide. Upon simple separation, the target protein or peptide with an authentic N‐terminus is then released in the solution by intein‐mediated cleavage. Different combinations of four self‐assembling peptides (ELK16, L₆KD, FK and FR) with three inteins (Sce VMA, Mtu ΔI‐CM and Ssp DnaB) were explored. One protein and two peptides were used as model polypeptides to test the strategy. The intein Mtu ΔI‐CM, which has pH‐shift inducible cleavage, was found to work well with three self‐assembling peptides (L₆KD, FR, FK). Using this intein gave a yield of protein or peptide comparable with that from other more established strategies, such as the Trx‐strategy, but in a simpler and more economical way. This strategy provides a simple and efficient method by which to prepare proteins and peptides with an authentic N‐terminus, which is especially effective for peptides of 30‐100 amino acids in length that are typically unstable and susceptible to degradation in Escherichia coli.     

High‐yield recombinant expression of the chicken antimicrobial peptide fowlicidin‐2 in Escherichia coli

The antimicrobial peptide fowlicidin‐2 identified in chicken is a member of the cathelicidins family. The mature fowlicidin‐2 possesses high antibacterial efficacy and lipopolysaccharide (LPS) neutralizing activity, and also represents an excellent candidate as an antimicrobial agent. In the present study, the recombinant fowlicidin‐2 was successfully produced by Escherichia coli (E. coli) recombinant expression system. The gene encoding fowlicidin‐2 with the codon preference of E. coli was designed through codon optimization and synthesized in vitro. The gene was then ligated into the plasmid pET‐32a(+), which features fusion protein thioredoxin at the N‐terminal. The recombinant plasmid was transformed into E. coli BL21(DE3) and cultured in Luria‐Bertani (LB) medium. After isopropyl‐β‐D‐thiogalactopyranoside (IPTG) induction, the fowlicidin‐2 fusion protein was successfully expressed as inclusion bodies. The inclusion bodies were dissolved and successfully released the peptide in 70% formic acid solution containing cyanogen bromide (CNBr) in a single step. After purification by reverse‐phase high‐performance liquid chromatography (RP‐HPLC), ～6.0 mg of fowlicidin‐2 with purity more than 97% was obtained from 1 litre of bacteria culture. The recombinant peptide exhibited high antibacterial activity against the Gram‐positive and Gram‐negative bacteria, and even drug‐resistant strains. This system could be used to rapidly and efficiently produce milligram quantities of a battery of recombinant antimicrobial peptides as well as for large‐scale production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:369–374, 2015     

Bone marrow mesenchymal stem cells increase motility of prostate cancer cells via production of stromal cell‐derived factor‐1α

Prostate cancer frequently metastasizes to the bone, and the interaction between cancer cells and bone microenvironment has proven to be crucial in the establishment of new metastases. Bone marrow mesenchymal stem cells (BM‐MSCs) secrete various cytokines that can regulate the behaviour of neighbouring cell. However, little is known about the role of BM‐MSCs in influencing the migration and the invasion of prostate cancer cells. We hypothesize that the stromal cell‐derived factor‐1α released by BM‐MSCs may play a pivotal role in these processes. To study the interaction between factors secreted by BM‐MSCs and prostate cancer cells we established an in vitro model of transwell co‐culture of BM‐MSCs and prostate cancer cells DU145. Using this model, we have shown that BM‐MSCs produce soluble factors which increase the motility of prostate cancer cells DU145. Neutralization of stromal cell‐derived factor‐1α (SDF1α) via a blocking antibody significantly limits the chemoattractive effect of bone marrow MSCs. Moreover, soluble factors produced by BM‐MSCs greatly activate prosurvival kinases, namely AKT and ERK 1/2. We provide further evidence that SDF1α is involved in the interaction between prostate cancer cells and BM‐MSCs. Such interaction may play an important role in the migration and the invasion of prostate cancer cells within bone.     

Bioproduction and characterization of a pH responsive self‐assembling peptide

Peptide P₁₁‐4 (QQRFEWEFEQQ) was designed to self‐assemble to form β‐sheets and nematic gels in the pH range 5–7 at concentrations ≥12.6 mM in water. This self‐assembly is reversibly controlled by adjusting the pH of the solvent. It can also self‐assemble into gels in biological media. This together with its biocompatibility and biodegradability make P₁₁‐4 an attractive building block for the fabrication of nanoscale materials with uses in, for example, tissue engineering. A limitation to large‐scale production of such peptides is the high cost of solid phase chemical synthesis. We describe expression of peptide P₁₁‐4 in the bacterium Escherichia coli from constructs carrying tandem repeats of the peptide coding sequence. The vector pET31b+ was used to express P₁₁‐4 repeats fused to the ketosteroid isomerase protein which accumulates in easily recoverable inclusion bodies. Importantly, the use of auto‐induction growth medium to enhance cell density and protein expression levels resulted in recovery of 2.5 g fusion protein/L culture in both shake flask and batch fermentation. Whole cell detergent lysis allowed recovery of inclusion bodies largely composed of the fusion protein. Cyanogen bromide cleavage followed by reverse phase HPLC allowed purification of the recombinant peptide with a C‐terminal homoserine lactone (rP₁₁‐4(hsl)). This recombinant peptide formed pH dependent hydrogels, displayed β‐structure measured by circular dichroism and fibril formation observed by transmission electron microscopy. Biotechnol. Bioeng. 2009;103: 241–251. © 2009 Wiley Periodicals, Inc.     

Production of an anti‐Candida peptide via fed batch and ion exchange chromatography

Interest in peptides as diagnostic and therapeutic materials require their manufacture via either a recombinant or synthetic route. This study examined the former, where a recombinant fusion consisting of an antifungal peptide was expressed and isolated from Escherichia coli. Fed batch fermentation with E. coli harboring an arabinose‐inducible plasmid produced the 12 residue anti‐Candida peptide fused to the N‐terminal of Green Fluorescent Protein (GFP_UV). The purification of the fusion protein, using ion‐exchange chromatography, was monitored by using the intrinsic fluorescence of GFP_UV. The recombinant antifungal peptide was successfully released by cyanogen bromide‐induced cleavage of the fusion protein. The recombinant peptide showed the expected antifungal activity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:865–871, 2016     

Self‐assembling organo‐peptide bolaphiles with KLK tripeptide head groups display selective antibacterial activity

In keeping with recent efforts to generate compounds for antibiotic and microbicide development, we focused on the creation of non‐natural organo‐peptide hybrids of antimicrobial peptide amides (KLK(L)_nKLK‐NH₂) derived from sapecin B and a self‐assembling oligoglycine organo‐peptide bolaphile containing an ω‐amino fatty acid residue. The hybrid organo‐peptide bolaphiles with two cationic KLK tripeptide motifs linked with an ω‐amino acid residue (penta‐, octa‐ or undecamethylene chain) maintained the self‐assembling properties of the root oligoglycine bolaphile. Electron microscopy clearly revealed complex supramolecular architectures for both sapecin B‐derived peptides and the hybrid analogues. FT‐IR spectroscopy indicated that the supramolecular structures were composed primarily of β‐sheets. CD revealed that the hybrid bolaphiles did not share the same secondary structures as the sapecin B peptides in solution. However, although secondary structures of antimicrobial peptides are central in the activity, the organo‐peptide bolaphiles also retained the potent antimicrobial activity of the leader sapecin B‐derived peptide against both Gram‐positive and Gram‐negative bacteria. In general, the hybrids were more selective than the sapecin B peptides, as they displayed little or no appreciable haemolytic activity. The results obtained herald a new approach for the design of purpose‐built hybrid organo‐peptide bolaphiles. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Discovery and characterization of Coturnix chinensis avian β‐defensin 10, with broad antibacterial activity

A novel avian β‐defensin (AvBD), AvBD10, was discovered in the liver and bone marrow tissues from Chinese painted quail (Coturnix chinensis) in the present study. The complete nucleotide sequence of quail AvBD10 contains a 207‐bp open reading frame that encodes 68 amino acids. The quail AvBD10 was expressed widely in all the tissues from quails except the tongue, crop, breast muscle, and thymus and was highly expressed in the bone marrow. In contrast to the expression pattern of AvBD10 in tissues from quail, the chicken AvBD10 was expressed in all 21 tissues from the layer hens investigated, with a high level of expression in the kidney, lung, liver, bone marrow, and Harderian glands. Recombinant glutathione S‐transferase (GST)‐tagged AvBD10s of both quail and chicken were produced and purified by expression of the two cDNAs in Escherichia coli, respectively. In addition, peptide according to the respective AvBD10s sequence was synthesized, named synthetic AvBD10s. As expected, both recombinant GST‐tagged AvBD10s and synthetic AvBD10s of quail and chicken exhibited similar bactericidal properties against most bacteria, including Gram‐positive and Gram‐negative forms. However, no significant bactericidal activity was found for quail recombinant GST‐tagged AvBD10 against Salmonella choleraesuis or for chicken recombinant GST‐tagged AvBD10 against Proteus mirabilis. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions

In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three‐dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long‐term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform‐sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live‐dead assay, and real‐time RT‐PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell‐seeded scaffold product for applications in regenerative medicine.     

Facile and selective covalent grafting of an RGD‐peptide to electrospun scaffolds improves HUVEC adhesion

The development of a biomimetic surface able to promote endothelialization is fundamental in the search for blood vessel substitutes that prevent the formation of thrombi or hyperplasia. This study aims at investigating the effect of functionalization of poly‐ε‐caprolactone or poly(L‐lactic acid‐co‐?‐caprolactone) electrospun scaffolds with a photoreactive adhesive peptide. The designed peptide sequence contains four Gly‐Arg‐Gly‐Asp‐Ser‐Pro motifs per chain and a p‐azido‐Phe residue at each terminus. Different peptide densities on the scaffold surface were obtained by simply modifying the peptide concentration used in pretreatment of the scaffold before UV irradiation. Scaffolds of poly‐ε‐caprolactone embedded with adhesive peptides were produced to assess the importance of peptide covalent grafting. Our results show that the scaffolds functionalized with photoreactive peptides enhance adhesion at 24 h with a dose‐dependent effect and control the proliferation of human umbilical vein endothelial cells, whereas the inclusion of adhesive peptide in the electrospun matrices by embedding does not give satisfactory results. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Bone‐forming capacity of adult human nasal chondrocytes

Nasal chondrocytes (NC) derive from the same multipotent embryological segment that gives rise to the majority of the maxillofacial bone and have been reported to differentiate into osteoblast‐like cells in vitro. In this study, we assessed the capacity of adult human NC, appropriately primed towards hypertrophic or osteoblastic differentiation, to form bone tissue in vivo. Hypertrophic induction of NC‐based micromass pellets formed mineralized cartilaginous tissues rich in type X collagen, but upon implantation into subcutaneous pockets of nude mice remained avascular and reverted to stable hyaline‐cartilage. In the same ectopic environment, NC embedded into ceramic scaffolds and primed with osteogenic medium only sporadically formed intramembranous bone tissue. A clonal study could not demonstrate that the low bone formation efficiency was related to a possibly small proportion of cells competent to become fully functional osteoblasts. We next tested whether the cues present in an orthotopic environment could induce a more efficient direct osteoblastic transformation of NC. Using a nude rat calvarial defect model, we demonstrated that (i) NC directly participated in frank bone formation and (ii) the efficiency of survival and bone formation by NC was significantly higher than that of reference osteogenic cells, namely bone marrow‐derived mesenchymal stromal cells. This study provides a proof‐of‐principle that NC have the plasticity to convert into bone cells and thereby represent an easily available cell source to be further investigated for craniofacial bone regeneration.     

Membrane partitioning of peptide aggregates: coarse-grained molecular dynamics simulations

Abstract

Coarse-grained molecular dynamics (CGMD) simulation technique (MARTINI force field) is applied to monitor the aggregation of helical peptides representing the transmembrane sequence and its extension of bone marrow stromal cell antigen 2 (BST-2). One of the peptides is coupled with a protein transducing domain (PTD) of nine arginine residues (R9) at its N-terminal side as well as a peptide, pep11**, which has been shown to bind to human papilloma virus 16 (HPV16) E6 oncoprotein. A short hydrophobic stretch of the transmembrane domain (TMD) of BST-2 aggregates the fastest and inserts into a lipid membrane. An aggregate of R9-pep11** attaches to the membrane via simultaneous contact of many arginine residues. Monomers from the aggregates of the shortest of the hydrophobic TMDs dissolve into the opposing leaflet when the aggregate spans the bilayer. A ‘flipping’ of the individual monomeric peptides is not observed.

Communicated by Ramaswamy H. Sarma     

Synthetic peptides derived from an N‐terminal domain of the E2 protein of GB virus C in the study of GBV‐C/HIV‐1 co‐infection

Synthetic peptides derived from GB virus C (GBV‐C) have previously been studied in our group for the development of new systems capable of diagnosing diseases caused by this humanotropic virus. We also recently described specific peptide domains of the E2 envelop protein of GBV‐C that have the capacity to interfere with the HIV‐1 fusion peptide, produce a notable decrease in cellular membrane fusion, and perturb HIV‐1 infectivity in a dose‐dependent manner. The present work discloses the design and synthesis of both linear and cyclic branched peptides based on a previously reported N‐terminal sequence of the GBV‐C E2 protein. Immunoassays and cell–cell fusion assays were performed to evaluate their diagnostic value to detect anti‐GBV‐C antibodies in HIV‐1 patients, as well as their putative anti‐HIV‐1 activity as entry inhibitors. Our results showed that chemical modifications of the selected E2(7–26) linear peptide to afford cyclic architecture do not result in an enhanced inhibition of gp41 HIV‐1‐mediated cell–cell fusion nor improved sensitivity in the detection of GBV‐C antibodies in HIV‐1 co‐infected patients. Thus, the ELISA data reinforce the potential utility of linear versions of the E2(7–26) region for the development of new peptide‐based immunosensor devices for the detection of anti‐GBV‐C antibodies in HIV‐1 co‐infected patients. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

The effect of autologous bone marrow stromal cells differentiated on scaffolds for canine tibial bone reconstruction

Bone marrow contains mesenchymal stem cells that form many tissues. Various scaffolds are available for bone reconstruction by tissue engineering. Osteoblastic differentiated bone marrow stromal cells (BMSC) promote osteogenesis on scaffolds and stimulate bone regeneration. We investigated the use of cultured autologous BMSC on different scaffolds for healing defects in tibias of adult male canines. BMSC were isolated from canine humerus bone marrow, differentiated into osteoblasts in culture and loaded onto porous ceramic scaffolds including hydroxyapatite 1, hydroxyapatite gel and calcium phosphate. Osteoblast differentiation was verified by osteonectine and osteocalcine immunocytochemistry. The scaffolds with stromal cells were implanted in the tibial defect. Scaffolds without stromal cells were used as controls. Sections from the defects were processed for histological, ultrastructural, immunohistochemical and histomorphometric analyses to analyze the healing of the defects. BMSC were spread, allowed to proliferate and differentiate to osteoblasts as shown by alizarin red histochemistry, and osteocalcine and osteonectine immunostaining. Scanning electron microscopy showed that BMSC on the scaffolds were more active and adhesive to the calcium phosphate scaffold compared to the others. Macroscopic bone formation was observed in all groups, but scaffolds with stromal cells produced significantly better results. Bone healing occurred earlier and faster with stromal cells on the calcium phosphate scaffold and produced more callus compared to other scaffolds. Tissue healing and osteoblastic marker expression also were better with stromal cells on the scaffolds. Increased trabecula formation, cell density and decreased fibrosis were observed in the calcium phosphate scaffold with stromal cells. Autologous cultured stromal cells on the scaffolds were useful for healing of canine tibial bone defects. The calcium phosphate scaffold was the best for both cell differentiation in vitro and bone regeneration in vivo. It may be possible to improve healing of bone defects in humans using stem cells from bone marrow.     

Prokaryotic expression and antimicrobial mechanism of XPF‐St7‐derived α‐helical peptides

XPF‐St7 (GLLSNVAGLLKQFAKGGVNAVLNPK) is an antimicrobial peptide isolated from Silurana tropicalis. We developed an α‐helical segment of XPF‐St7 termed as XPF2. Using the XPF2 as a framework, we increased the positive net charge of XPF2 by amino acid substitutions, and thus obtained two novel antimicrobial peptides XPF4 and XPF6. These were each fused with an ubiquitin tag and successfully expressed in Escherichia coli. This ubiquitin fusion system may present a viable alternative for industrial production of antimicrobial peptides. XPF4 and XPF6 showed much better overall antimicrobial activity against both Gram‐negative and Gram‐positive bacteria than XPF2. The therapeutic index of XPF4 and XPF6 was 5.6‐fold and 6.7‐fold of XPF2, respectively. Bacterial cell membrane permeabilization and genomic DNA interaction assays were utilized to explore the mechanism of action of XPF serial peptides. The results revealed that the target of these antimicrobial peptides was the bacterial cytoplasmic membrane. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Growth and functional harvesting of human mesenchymal stromal cells cultured on a microcarrier‐based system

Human mesenchymal stromal cells (hMSCs) cells are attractive for applications in tissue engineering and cell therapy. Because of the low availability of hMSCs in tissues and the high doses of hMSCs necessary for infusion, scalable and cost‐effective technologies for in vitro cell expansion are needed to produce MSCs while maintaining their functional, immunophenotypic and cytogenetic characteristics. Microcarrier‐based culture systems are a good alternative to traditional systems for hMSC expansion. The aim of the present study was to develop a scalable bioprocess for the expansion of human bone marrow mesenchymal stromal cells (hBM‐MSCs) on microcarriers to optimize growth and functional harvesting. In general, the results obtained demonstrated the feasibility of expanding hBM‐MSCs using microcarrier technology. The maximum cell concentration (n = 5) was ～4.82 ± 1.18 × 10⁵ cell mL^?1 at day 7, representing a 3.9‐fold increase relative to the amount of inoculated cells. At the end of culture, 87.2% of the cells could be harvested (viability = 95%). Cell metabolism analysis revealed that there was no depletion of important nutrients such as glucose and glutamine during culture, and neither lactate nor ammonia byproducts were formed at inhibitory concentrations. The cells that were recovered after the expansion retained their immunophenotypic and functional characteristics. These results represent an important step toward the implementation of a GMP‐compliant large‐scale production system for hMSCs for cellular therapy. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:889–895, 2014     

Identification of peptide‐binding sites within BSA using rapid,laser‐induced covalent cross‐linking combined with high‐performance mass spectrometry

We are developing a rapid, time‐resolved method using laser‐activated cross‐linking to capture protein‐peptide interactions as a means to interrogate the interaction of serum proteins as delivery systems for peptides and other molecules. A model system was established to investigate the interactions between bovine serum albumin (BSA) and 2 peptides, the tridecapeptide budding‐yeast mating pheromone (α‐factor) and the decapeptide human gonadotropin‐releasing hormone (GnRH). Cross‐linking of α‐factor, using a biotinylated, photoactivatable p‐benzoyl‐L‐phenylalanine (Bpa)–modified analog, was energy‐dependent and achieved within seconds of laser irradiation. Protein blotting with an avidin probe was used to detect biotinylated species in the BSA‐peptide complex. The cross‐linked complex was trypsinized and then interrogated with nano‐LC–MS/MS to identify the peptide cross‐links. Cross‐linking was greatly facilitated by Bpa in the peptide, but some cross‐linking occurred at higher laser powers and high concentrations of a non‐Bpa–modified α‐factor. This was supported by experiments using GnRH, a peptide with sequence homology to α‐factor, which was likewise found to be cross‐linked to BSA by laser irradiation. Analysis of peptides in the mass spectra showed that the binding site for both α‐factor and GnRH was in the BSA pocket defined previously as the site for fatty acid binding. This model system validates the use of laser‐activation to facilitate cross‐linking of Bpa‐containing molecules to proteins. The rapid cross‐linking procedure and high performance of MS/MS to identify cross‐links provides a method to interrogate protein‐peptide interactions in a living cell in a time‐resolved manner.     

Fetal Liver Stromal Cells Support Blast Growth in Transient Abnormal Myelopoiesis in Down Syndrome Through GM‐CSF

Transient abnormal myelopoiesis (TAM) in neonates with Down syndrome, which spontaneously resolves within several weeks or months after birth, may represent a very special form of leukemia arising in the fetal liver (FL). To explore the role of the fetal hematopoietic microenvironment in the pathogenesis of TAM, we examined the in vitro influences of stromal cells of human FL and fetal bone marrow (FBM) on the growth of TAM blasts. Both FL and FBM stromal cells expressed mesenchymal cell antigens (vimentin, α‐smooth muscle actin, CD146, and nestin), being consistent with perivascular cells/mesenchymal stem cells that support hematopoietic stem cells. In addition, a small fraction of the FL stromal cells expressed an epithelial marker, cytokeratin 8, indicating that they could be cells in epithelial‐mesenchymal transition (EMT). In the coculture system, stromal cells of the FL, but not FBM, potently supported the growth of TAM blast progenitors, mainly through humoral factors. High concentrations of hematopoietic growth factors were detected in culture supernatants of the FL stromal cells and a neutralizing antibody against granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) almost completely inhibited the growth‐supportive activity of the culture supernatants. These results indicate that FL stromal cells with unique characteristics of EMT cells provide a pivotal hematopoietic microenvironment for TAM blasts and that GM‐CSF produced by FL stromal cells may play an important role in the pathogenesis of TAM. J. Cell. Biochem. 115: 1176–1186, 2014. © 2014 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals, Inc.     

Enhanced transport of plant‐produced rabies single‐chain antibody‐RVG peptide fusion protein across an in cellulo blood–brain barrier device

The biomedical applications of antibody engineering are developing rapidly and have been expanded to plant expression platforms. In this study, we have generated a novel antibody molecule in planta for targeted delivery across the blood–brain barrier (BBB). Rabies virus (RABV) is a neurotropic virus for which there is no effective treatment after entry into the central nervous system. This study investigated the use of a RABV glycoprotein peptide sequence to assist delivery of a rabies neutralizing single‐chain antibody (ScFv) across an in cellulo model of human BBB. The 29 amino acid rabies virus peptide (RVG) recognizes the nicotinic acetylcholine receptor (nAchR) at neuromuscular junctions and the BBB. ScFv and ScFv‐RVG fusion proteins were produced in Nicotiana benthamiana by transient expression. Both molecules were successfully expressed and purified, but the ScFv expression level was significantly higher than that of ScFv‐RVG fusion. Both ScFv and ScFv‐RVG fusion molecules had potent neutralization activity against RABVin cellulo. The ScFv‐RVG fusion demonstrated increased binding to nAchR and entry into neuronal cells, compared to ScFv alone. Additionally, a human brain endothelial cell line BBB model was used to demonstrate that plant‐produced ScFv‐RVG^P fusion could translocate across the cells. This study indicates that the plant‐produced ScFv‐RVG^P fusion protein was able to cross the in celluloBBB and neutralize RABV.