Gene biomarkers and classifiers for various subtypes of HTLV-1-caused ATLL cancer identified by a combination of differential gene co­expression and support vector machine algorithms.

Adult T-cell leukemia/lymphoma (ATLL) is pathogen-caused cancer that is progressed after the infection by human T-cell leukemia virus type 1. Four significant subtypes comprising acute, lymphoma, chronic, and smoldering have been identified for this cancer. However, there are no trustworthy prognostic biomarkers for these subtypes. We utilized a combination of two powerful network-based and machine-learning algorithms including differential co-expressed genes (DiffCoEx) and support vector machine-recursive feature elimination with cross-validation (SVM-RFECV) methods to categorize disparate ATLL subtypes from asymptomatic carriers (ACs). The results disclosed the significant involvement of CBX6, CNKSR1, and MAX in chronic, MYH10 and P2RY1 in acute, C22orf46 and HNRNPA0 in smoldering subtypes. These genes also can classify each ATLL subtype from AC carriers. The integration of the results of two powerful algorithms led to the identification of reliable gene classifiers and biomarkers for diverse ATLL subtypes.

Exploration of mRNAs and miRNA classifiers for various ATLL cancer subtypes using machine learning.

BACKGROUND: Adult T-cell Leukemia/Lymphoma (ATLL) is a cancer disease that is developed due to the infection by human T-cell leukemia virus type 1. It can be classified into four main subtypes including, acute, chronic, smoldering, and lymphoma. Despite the clinical manifestations, there are no reliable diagnostic biomarkers for the classification of these subtypes. METHODS: Herein, we employed a machine learning approach, namely, Support Vector Machine-Recursive Feature Elimination with Cross-Validation (SVM-RFECV) to classify the different ATLL subtypes from Asymptomatic Carriers (ACs). The expression values of multiple mRNAs and miRNAs were used as the features. Afterward, the reliable miRNA-mRNA interactions for each subtype were identified through exploring the experimentally validated-target genes of miRNAs. RESULTS: The results revealed that miR-21 and its interactions with DAAM1 and E2F2 in acute, SMAD7 in chronic, MYEF2 and PARP1 in smoldering subtypes could significantly classify the diverse subtypes. CONCLUSIONS: Considering the high accuracy of the constructed model, the identified mRNAs and miRNA are proposed as the potential therapeutic targets and the prognostic biomarkers for various ATLL subtypes.

Decoding pathogenesis factors involved in the progression of ATLL or HAM/TSP after infection by HTLV-1 through a systems virology study.

BACKGROUND: Human T-cell Leukemia Virus type-1 (HTLV-1) is a retrovirus that causes two diseases including Adult T-cell Leukemia/Lymphoma (ATLL cancer) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP, a neurodegenerative disease) after a long latency period as an asymptomatic carrier (AC). There are no obvious explanations about how each of the mentioned diseases develops in the AC carriers. Finding the discriminative molecular factors and pathways may clarify the destiny of the infection. METHODS: To shed light on the involved molecular players and activated pathways in each state, differentially co-expressed modules (DiffCoEx) algorithm was employed to identify the highly correlated genes which were co-expressed differently between normal and ACs, ACs and ATLL, as well as ACs and HAM/TSP samples. Through differential pathway analysis, the dysregulated pathways and the specific disease-genes-pathways were figured out. Moreover, the common genes between the member of DiffCoEx and differentially expressed genes were found and the specific genes in ATLL and HAM/TSP were introduced as possible biomarkers. RESULTS: The dysregulated genes in the ATLL were mostly enriched in immune and cancer-related pathways while the ones in the HAM/TSP were enriched in immune, inflammation, and neurological pathways. The differential pathway analysis clarified the differences between the gene players in the common activated pathways. Eventually, the final analysis revealed the involvement of specific dysregulated genes including KIRREL2, RAB36, and KANK1 in HAM/TSP as well as LTB4R2, HCN4, FZD9, GRIK5, CREB3L4, TACR2, FRMD1, LHB, FGF3, TEAD3, GRIN2D, GNRH2, PRLH, GPR156, and CRHR2 in ATLL. CONCLUSION: The identified potential prognostic biomarkers and therapeutic targets are proposed as the most important platers in developing ATLL or HAM/TSP. Moreover, the proposed signaling network clarifies the differences between the functional players in the activated pathways in ACs, ATLL, and HAM/TSP.

Lactococcus-based vaccine against brucellosis: IgG immune response in mice with rOmp16-IL2 fusion protein.

This study was designed to introduce the recombinant Lactococcus lactis MG1363 as a cell factory candidate for production of recombinant Brucella melitensis Omp16-Human IL2 (r-Omp16-IL2) and to suggest it as a promising safe, non-pathogenic mucosal live vaccine against brucellosis. Three groups of BALB/c mice (10 mice per group) were intragastrically administrated with phosphate-buffered saline (PBS), L. lactis harboring the empty pAMJ2008 plasmid and with L. lactis expressing rOmp-IL2. The first two groups were classified as control groups and the third one is indicated as treatment group. Another group was injected by the intraperitoneal (i.p.) route with purified rOmp16-IL2 protein. The total serum IgG of each group was assessed with indirect ELISAs at two days before immunization and also two weeks after the last immunization. Results showed that BALB/c mice intragastrically administrated with L. lactis expressing rOmp-IL2 had dominant IgG response compared to the control (PBS administrated) group (P < 0.05). The level of IgG was significantly increased by intraperitoneally injection of recombinant Omp-IL2 in adjuvant compared to the intragastrically administration of PBS and L. lactis/pAMJ2008 as control groups, and also compared to L. lactis/pAMJ2008-rOmp-IL2 (P < 0.05). Our findings provide the use of L. lactis rOmp16-IL2 as a new promising alternative safe strategy than presently live attenuated vaccines toward developing an oral vaccine or subunit-based vaccine against brucellosis.

Production of Brucella melitensis Omp16 protein fused to the human interleukin 2 in Lactococcus lactis MG1363 toward developing a Lactococcus-based vaccine against brucellosis.

The use of the food-grade bacterium Lactococcus lactis as a new cell factory is a promising alternative expression system for producing a desired protein. The Omp16-IL2 fusion protein antigen was cloned, expressed, and purified in this study. The Omp16-IL2 fusion gene was designed and cloned in pGH plasmid with appropriate restriction sites and subcloned in pAMJ2008 expression vector digested with the same enzymes. The purified recombinant constructed pAMJ-rOmp-IL2 was introduced into L. lactis subsp. cremoris MG1363 by electrotransformation. Finally, the expression and purification of Omp16-IL2 fusion protein was investigated. This study reports the construction of a recombinant L. lactis expressing the Omp16-IL2 fusion protein as an oral Lactococcus-based vaccine, as compared with commonly used live attenuated vaccines, for future studies against brucellosis.

Expression of Helicobacter pylori CagL gene in Lactococcus lactis MG1363 and evaluation of its immunogenicity as an oral vaccine in mice.

Helicobacter pylori is a gram negative pathogen which commonly colonizes in the human gastric mucosa from early childhood and persists throughout life. CagL is a 27-kDa protein that is located at the tip of T4SS pili and highly conserved among pathogenic H. pylori strains. Lactic acid bacteria especially Lactococcus lactis (L. lactis) could serve as an antigen-delivering vehicle for the development of edible vaccine. In this study H. pylori CagL gene was cloned in pAMJ2008 vector and transferred to Lactococcus lactis MG1363 as the host for CagL antigen production. This recombinant bacterium was orally subjected to mice, and the immune response to CagL was evaluated by ELISA. Intracellular expression of CagL protein was confirmed by Western blot analysis. Mucosal immunization of mice with the recombinant L. lactis significantly stimulated CagL-Specific antibodies: IgA, IgG, cytokine IL-17 and IFN-γ. Moreover, the specific anti-CagL IgA response was detected in the feces of immunized mice. These results indicate that CagL of H. pylori was successfully expressed in L. lactis and the recombinant bacteria can be potentially used as an edible vaccine against H. pylori infection.

New insights into the molecular characteristics behind the function of Renilla luciferase.

Renilla Luciferase (RLuc) is a blue light emitter protein which can be applied as a valuable tool in medical diagnosis. But due to lack of the crystal structure of RLuc-ligand complex, the functional motions and catalytic mechanism of this enzyme remain largely unknown. In the present study, the active site properties and the ligand-receptor interactions of the native RLuc and its red-shifted light emitting variant (Super RLuc 8) were investigated using molecular docking approach, molecular dynamics (MD) analysis, and MM-PBSA method. The detailed analysis of the main clusters led to identifying a lid-like structure and its functional motions. Furthermore, an induced-fit mechanism is proposed where ligand-binding induces conformational changes of the active site. Our findings give an insight into the deeper understanding of RLuc conformational changes during binding steps and ligand-receptor pattern. Moreover, our work broaden our understanding of how active site geometry is adjusted to support the catalytic activity and red-shifted light emission in Super RLuc 8.

Molecular basis of thermostability enhancement of Renilla luciferase at higher temperatures by insertion of a disulfide bridge into the structure.

Renilla luciferase (RLuc), also known as Renilla-luciferin 2-monooxygenase, is a light producing enzyme used in many biotechnological applications such as bioreporters. However, its kinetics stability -especially at higher temperatures- is a limiting factor for developing thermostable bioreporters. The aim of this study was to improve the stability of super Renilla luciferase 8 (SRLuc 8) which is a red-emitter variety of RLuc at higher temperatures, by introduction of a disulfide bridge into its structure. In this study, the choice of the proper disulfide bond formation was based on computational methods and enzyme functionality (active site position) which is called geometric-functional method. N45 and A71 at the N-terminal of the enzyme were selected for directed evolution. The engineered luciferase was called C-SRLuc 8 and its activity and stability were assayed. The results indicated that the kinetic stability of C-SRLuc 8 increased significantly at 60°C to 70°C as compared to SRLuc 8; the residual activity of C-SRLuc 8 was approximately 20% after incubation at 65°C for 5min. Moreover, the enzyme activity decreased compared with SRLuc 8. The molecular basis of the structural changes was considered using molecular dynamics simulations and the results indicated that the N45C/A71C crosslink was involved in a hotspot foldon which seemed to be the rate-limiting step of conformational collapse at higher temperatures. The present study may provide an opportunity for the development of the next-generation of thermostable RLuc-based biosensors.

Optimized protocol for soluble prokaryotic expression, purification and structural analysis of human placenta specific-1(PLAC1).

Placenta specific -1 (PLAC1) has been recently introduced as a small membrane-associated protein mainly involved in placental development. Expression of PLAC1 transcript has been documented in almost one hundred cancer cell lines standing for fourteen distinct cancer types. The presence of two disulfide bridges makes difficult to produce functional recombinant PLAC1 in soluble form with high yield. This limitation also complicates the structural studies of PLAC1, which is important for prediction of its physiological roles. To address this issue, we employed an expression matrix consisting of two expression vectors, five different E. coli hosts and five solubilization conditions to optimize production of full and truncated forms of human PLAC1. The recombinant proteins were then characterized using an anti-PLAC1-specific antibody in Western blotting (WB) and enzyme linked immunosorbent assay (ELISA). Structure of full length protein was also investigated using circular dichroism (CD). We demonstrated the combination of Origami™ and pCold expression vector to yield substantial amount of soluble truncated PLAC1 without further need for solubilization step. Full length PLAC1, however, expressed mostly as inclusion bodies with higher yield in Origami™ and Rosetta2. Among solubilization buffers examined, buffer containing Urea 2 M, pH 12 was found to be more effective. Recombinant proteins exhibited excellent reactivity as detected by ELISA and WB. The secondary structure of full length PLAC1 was considered by CD spectroscopy. Taken together, we introduced here a simple, affordable and efficient expression system for soluble PLAC1 production.

Improvement of erythrose reductase activity, deletion of by-products and statistical media optimization for enhanced erythritol production from Yarrowia lipolytica mutant 49.

The purpose of the present investigation was to produce erythritol by Yarrowia lipolytica mutant without any by-products. Mutants of Y. lipolytica were generated by ultra-violet for enhancing erythrose reductase (ER) activity and erythritol production. The mutants showing the highest ER activity were screened by triphenyl tetrazolium chloride agar plate assay. Productivity of samples was analyzed by thin-layer chromatography and high-performance liquid chromatography equipped with the refractive index detector. One of the mutants named as mutant 49 gave maximum erythritol production without any other by-products (particularly glycerol). Erythritol production and specific ER activity in mutant 49 increased to 1.65 and 1.47 times, respectively, in comparison with wild-type strain. The ER gene of wild and mutant strains was sequenced and analyzed. A general comparison of wild and mutant gene sequences showed the replacement of Asp(270) with Glu(270) in ER protein. In order to enhance erythritol production, we used a three component-three level-one response Box-Behnken of response surface methodology model. The optimum medium composition for erythritol production was found to be (g/l) glucose 279.49, ammonium sulfate 9.28, and pH 5.41 with 39.76 erythritol production.

Exploring the Potential of Arginine to Increase Coelenterazine-Renilla Luciferase Affinity and Enzyme Stability: Kinetic and Molecular Dynamics Studies.

Renilla luciferase catalyzes the oxidation of coelenterazine to coelenteramide and results in the emission of a photon of light. Although Renilla luciferase has various applications in biotechnology, its low thermal stability limits the development of its applications. Arginine is a well-known stabilizing amino acid that plays a key role in protein stabilization against inactivation. However, its impact on enzyme properties is unpredictable. This study investigates the impact of arginine on the kinetics and thermal stability of Renilla luciferase. The enzyme's performance was significantly enhanced in the presence of arginine, with catalytic efficiency increasing by 3.31-fold and 3.08-fold when exposed to 0.2 M and 0.3 M arginine, respectively. Additionally, arginine improved the thermal stability of Renilla luciferase. Molecular dynamics simulation showed that the addition of 0.2 M arginine reduced the binding of coelenteramide, the reaction product and an enzyme inhibitor, to the active site of the Renilla luciferase. Therefore, the release of the product was accelerated, and the affinity of Renilla luciferase for coelenterazine increased. Furthermore, Molecular dynamics studies indicated an increased network of water molecules surrounding Renilla luciferase in the presence of 0.2 M arginine. This network potentially enhances the hydrophobic effect on the protein structure, ultimately improving enzyme stability. The findings of this study hold promise for the development of commercial kits incorporating Renilla luciferase.

Development of a bioluminescent homogenous nanobody-based immunoassay for the detection of prostate-specific antigen (PSA).

Prostate cancer is the most prevalent cancer in men. At present, the diagnosis and screening of prostate cancer rely on the essential biomarker known as prostate-specific antigen (PSA). The main purpose of this study was to develop a novel immunoassay for the detection of PSA based on a tri-part split-nanoluciferase system and a nanobody targeting PSA. In our approach, two small components of the split-nanoluciferase, referred to as ß9 and ß10, were individually fused to two anti-PSA nanobodies, N7 and N23. When these proteins bind to PSA and in the presence of the third nanoluciferase component, called Δ11S, the split-nanoluciferase components are brought into close proximity, facilitating the reassembly of the active nanoluciferase and activation of luminescence. These proteins were expressed in a bacterial expression system, purified, and employed for the intended immunoassay. The developed immunoassay demonstrated the capability to sensitively detect PSA within a linear range from 1.0 to 20.0 ng/mL with LOD of 0.4 ng/mL, and the results obtained through this immunoassay agreed with those derived from the ELISA. Our study indicates that the homogeneous immunoassay developed with nanobodies exhibits remarkable specificity for PSA and can serve as a reliable, fast, and user-friendly test for detecting PSA.

Uncovering the role of sorbitol in Renilla luciferase kinetics: Insights from spectroscopic and molecular dynamics studies.

Renilla luciferase catalyzes the oxidation of coelenterazine to coelenteramide, resulting in the emission of a photon of light. This study investigated the impact of sorbitol on the structural and kinetic properties of Renilla luciferase using circular dichroism, fluorescence spectroscopy, and molecular dynamics simulations. Our investigation, carried out using circular dichroism and fluorescence analyses, as well as a thermal stability assay, has revealed that sorbitol induces conformational changes in the enzyme but does not improve its thermal stability. Moreover, through kinetic studies, it has been demonstrated that at a concentration of 0.4 M, sorbitol enhances the catalytic efficiency of Renilla luciferase. However, at higher concentrations, sorbitol results in a decrease in catalytic efficiency. Additionally, molecular dynamics simulations have shown that sorbitol increases the presence of hydrophobic pockets on the enzyme's surface. These simulations have also provided evidence that at a concentration of 0.4 M, sorbitol facilitates substrate access to the active site of the enzyme. Nevertheless, at higher concentrations, sorbitol obstructs substrate trafficking, most likely due to its impact on the gateway to the active site. This study may provide insights into the kinetic changes observed in enzymes with buried active sites, such as those with α/ß hydrolase fold.

Enhancing protein delivery for tissue regeneration: Development of AGR2-loaded hydrogels with controlled release properties.

The growth factor Anterior Gradient 2 (AGR2) has been shown to have an effective role in tissue regeneration, but remained largely unexplored in localized tissue engineering applications. Alginate beads have been proven as safe carriers for protein encapsulation, but they suffer from fragility and uncontrolled protein release. For such alginate systems, little is known about how changes in concentrations and ion-crosslinking affect protein release and accumulation in 3-D matrices. To address these questions, an engineered interpenetrating polymer network (IPN) has been used to synthesize a novel hybrid system consisting of AGR2 loaded beads composed of calcium-crosslinked sodium alginate (SA) and carboxymethyl cellulose (CMC). These beads are embedded in films consisting of SA and polyvinyl alcohol (PVA), using a simple ion gelation technique. We assess protein release kinetics and accumulation within the hybrid system by varying polymer concentrations and cross-linking parameters. The IPN hybrid system maintains controlled release over two weeks, without an initial burst period. Through this approach efficicnt delivery of AGR2 is achieved which in turn effectively mediates cell migration and proliferation, resulting in excellent cell viability and complete wound closure. The described release system opens new perspectives in tissue engineering.

Tri-part NanoLuc as a new split technology with potential applications in chemical biology: a mini-review.

For several decades, researchers have been using protein-fragment complementation assay (PCA) approaches for biosensing to study protein-protein interaction for a variety of aims, including viral infection, cellular apoptosis, G protein coupled receptor (GPCR) signaling, drug and substrate screening, and protein aggregation and protein editing by CRISPR/Cas9. As a reporter, NanoLuc (NLuc), a smaller and the brightest engineered luciferase derived from deep-sea shrimp Oplophorus gracilirostris, has been found to have many benefits over other luminescent enzymes in PCA. Inspired by the split green fluorescent protein (GFP) and its ß-barrel structure, two split NLuc consisting of peptide fragments have been reported including the binary and ternary NLuc systems. NanoBiT® (large fragment + peptide) has been used extensively. In contrast, tripart split NLuc (large fragment + 2 peptides) has been applied and hardly used, while it has some advantages over NanoBiT in some studies. Nevertheless, tripart NLuc has some drawbacks and challenges to overcome but has several potential characteristics to become a multifunctional and powerful tool. In this review, several aspects of tripart NLuc are studied and a brief comparison with NanoBiT® is given.

Potential miRNA-gene interactions determining progression of various ATLL cancer subtypes after infection by HTLV-1 oncovirus.

BACKGROUND: Adult T-cell Leukemia/Lymphoma (ATLL) is a rapidly progressing type of T-cell non-Hodgkin lymphoma that is developed after the infection by human T-cell leukemia virus type 1 (HTLV-1). It could be categorized into four major subtypes, acute, lymphoma, chronic, and smoldering. These different subtypes have some shared clinical manifestations, and there are no trustworthy biomarkers for diagnosis of them. METHODS: We applied weighted-gene co-expression network analysis to find the potential gene and miRNA biomarkers for various ATLL subtypes. Afterward, we found reliable miRNA-gene interactions by identifying the experimentally validated-target genes of miRNAs. RESULTS: The outcomes disclosed the interactions of miR-29b-2-5p and miR-342-3p with LSAMP in ATLL_acute, miR-575 with UBN2, miR-342-3p with ZNF280B, and miR-342-5p with FOXRED2 in ATLL_chronic, miR-940 and miR-423-3p with C6orf141, miR-940 and miR-1225-3p with CDCP1, and miR-324-3p with COL14A1 in ATLL_smoldering. These miRNA-gene interactions determine the molecular factors involved in the pathogenesis of each ATLL subtype and the unique ones could be considered biomarkers. CONCLUSION: The above-mentioned miRNAs-genes interactions are suggested as diagnostic biomarkers for different ATLL subtypes.

Renilla luciferase-labeled Annexin V: a new probe for detection of apoptotic cells.

The Ca(2+)-dependent binding of Annexin V to phosphatidylserine on cell surfaces is a reliable marker for apoptosis that is widely used in flow cytometry based apoptosis assays. In this paper, we report a new class of Annexin V-based probes for apoptosis. Luciferase from Renilla reniformis (RLuc) was linked to Annexin V and expressed successfully in a soluble form in Escherichia coli BL21 (DE3). The new probe, Rluc/Annexin V, was purified and functionally assayed for detection of apoptosis in actinomycin D-induced apoptotic Jurkat cells. Moreover, the spontaneous apoptosis in neutrophils was shown using the new probe. The results indicate that Rluc/Annexin V can bind to the apoptotic cells, and the signal of Renilla luciferase can be detected by luminometric measurements. The availability of Rluc/Annexin V may be of potential commercial interest for improving current apoptosis assays.

Integration of gene co-expression analysis and multi-class SVM specifies the functional players involved in determining the fate of HTLV-1 infection toward the development of cancer (ATLL) or neurological disorder (HAM/TSP).

Human T-cell Leukemia Virus type-1 (HTLV-1) is an oncovirus that may cause two main life-threatening diseases including a cancer type named Adult T-cell Leukemia/Lymphoma (ATLL) and a neurological and immune disturbance known as HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). However, a large number of the infected subjects remain as asymptomatic carriers (ACs). There is no comprehensive study that determines which dysregulated genes differentiate the pathogenesis routes toward ATLL or HAM/TSP. Therefore, two main algorithms including weighted gene co-expression analysis (WGCNA) and multi-class support vector machines (SVM) were utilized to find major gene players in each condition. WGCNA was used to find the highly co-regulated genes and multi-class SVM was employed to identify the most important classifier genes. The identified modules from WGCNA were validated in the external datasets. Furthermore, to find specific modules for ATLL and HAM/TSP, the non-preserved modules in another condition were found. In the next step, a model was constructed by multi-class SVM. The results revealed 467, 3249, and 716 classifiers for ACs, ATLL, and HAM/TSP, respectively. Eventually, the common genes between the WGCNA results and classifier genes resulted from multi-class SVM that also determined as differentially expressed genes, were identified. Through these step-wise analyses, PAIP1, BCAS2, COPS2, CTNNB1, FASLG, GTPBP1, HNRNPA1, RBBP6, TOP1, SLC9A1, JMY, PABPC3, and PBX1 were found as the possible critical genes involved in the progression of ATLL. Moreover, FBXO9, ZNF526, ERCC8, WDR5, and XRCC3 were identified as the conceivable major involved genes in the development of HAM/TSP. These genes can be proposed as specific biomarker candidates and therapeutic targets for each disease.

Design and production of a novel chimeric human growth hormone superagonist fused to human Fc domain.

Background and purpose: Growth hormone (GH) has been known as a crucial metabolic hormone expressed at the pituitary and the other number of cells and tissues and responsible for body growth. Because of the short half-life of GH, daily subcutaneous injections were shown to be more effective for GH therapy. This represents a burden for patients. So, there is a strong effort from the industry to create a long-acting form of GH and lots of technologies like GH fusion proteins are used to increase GH half-life. Experimental approach: In this study, the Fc domain of human IgG1 with serine-glycine linkers was attached to the C-terminal of a GH superagonist via molecular cloning. The presence of recombinant vector in E. coli host was confirmed by PCR. SDS-PAGE and western blot analysis showed the expression of recombinant proteins in the bacterial lysate. The binding ability to growth hormone receptors is determined by ELISA. Findings / Results: Our results showed that the novel SupGH-Fc has a good binding affinity to its receptor in ELISA in comparison to standard GH, although it has a big size. Conclusion and implications: Our data in this study clearly demonstrated the expression of the SupGH-Fc in a recombinant protein expression system. It is an introduction to the production of the new recombinant GH, which can bind to its receptor more effectively than commercial growth hormones and also might have a longer half-life.

A recombinant affitoxin derived from a HER3 affibody and diphteria-toxin has potent and selective antitumor activity.

High expression of receptor tyrosine-protein kinase erbB-3 (HER3) has been found in several malignancies such as breast cancer. In this study, we designed, produced and evaluated a new affitoxin consisting of a truncated form of diphtheria toxin and a HER3-binding affibody domains. The new affitoxin was expressed in Escherichia coli and purified by affinity chromatography. We evaluated the suitability of affitoxin to kill HER3 positive breast cancer cells with MTT and apoptosis assays. The protein synthesis inhibition was also evaluated. The IC50 value in HER3 negative cells is about 10 times more than HER3 positive cells in new design of affitoxin. The specificity of affitoxin for binding to HER3 positive cells was also investigated with binding assay with flow cytometry. The results show that, the new affitoxin is an anti-cancer molecule with specific binding to HER3 positive cells and may open a new window for the treatment of HER3-positive cancers.