Production of antimicrobial peptide (Oxysterlin 1) in Escherichia coli with ELP self-cleavage tag / 生物工程学报

Antimicrobial peptides are the most promising alternatives to antibiotics. However, the strategy of producing antimicrobial peptides by recombinant technology is complicated and expensive, which is not conducive to the large-scale production. Oxysterlin 1 is a novel type of cecropin antimicrobial peptide mainly targeting on Gram-negative bacteria and is of low cytotoxicity. In this study, a simple and cost-effective method was developed to produce Oxysterlin 1 in Escherichia coli. The Oxysterlin 1 gene was cloned into a plasmid containing elastin-like polypeptide (ELP) and protein splicing elements (intein) to construct the recombinant expression plasmid (pET-ELP-I-Oxysterlin 1). The recombinant protein was mainly expressed in soluble form in E. coli, and then the target peptide can be purified with a simple salting out method followed by pH changing. The final yield of Oxysterlin 1 was about 1.2 mg/L, and the subsequent antimicrobial experiment showed the expected antimicrobial activity. This study holds promise for large-scale production of antimicrobial peptides and the in-depth study of its antimicrobial mechanism.

Construction of spider silk functional platform via intein trans-splicing / 生物工程学报

To provide technical support for spider silk functional modification, we developed a simple and efficient functional platform via intein trans-splicing. Small ubiquitin-related modifier protein (SUMO) was fused to the recombinant spider silk protein (W2CT) by peptide bond via S0 split intein Ssp DnaB trans-splicing, resulting in a protein SUMOW2CT. However, incorporation of exogenous protein led to mechanical property defect and lower fiber yield, and also slowed down the fiber assembly velocity but no obvious differences in supercontraction and chemical resistance when compared with fibers from W2CT (W). SUMO protease digestion showed positive results on the fibers, indicating that the SUMO protein kept its native conformation and bioactive. Above all, this work provides a technical support for spider silk high simply and efficient functionalized modification.

Application of inteins in building protein affinity purification system / 生物工程学报

Intein is a part of polypeptide in the premature protein with the capability of self-splicing, which is widely applied in protein purification, protein conjuction, cyclopeptide preparation, protein labeling and biosensor. In this review, we summarized the development of intein used in protein purification, discussed intein-mediated chromatographic and non-chromatographic purification systems, and summarized the researches in manipulating intein cleavage reaction. This work is to provide clues for improvement of intein-mediated protein purification.

Intein-mediated expression of cecropin in escherichia coli

Different strategies have been used to overcome the difficulties to produce antimicrobial peptides. Here we used Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT-System, New England Biolabs) for the expression of the antimicrobial peptide cecropin to reduce its sensitivity to intracellular proteases and use its inducible self-cleaving capability to remove the carrier. Cecropin was cloned into suitable expression vector pTYB11, and expression induced by IPTG in Escherichia coli ER2566. The use of 22ºC induction allowed the expression of cecropin with its intein carrier in soluble form. Cell extracts were purified by chitin affinity chromatography and intein-mediated splicing of the target protein was achieved by thiol addition, obtaining a final yield of 2.5 mg cecropin/l. Cecropin cleaved from the intein had its proper biologically active form, showing a micromolar antimicrobial activity against Vibrio ordalii, Vibrio alginolyticus and Escherichia coli.

An association study of single nucleotide polymorphisms of the FOXP3 intron-1 and the risk of Psoriasis vulgaris.

Psoriasis vulgaris (PV) is a common autoimmune disease that involves the dysfunction of CD4+CD25+ regulatory T cells. FOXP3 is a key transcription factor in the development and function of CD4+CD25+ regulatory T cells. Previous studies have demonstrated a genetic association between the FOXP3 gene and some autoimmune diseases. To elucidate the association between the FOXP3 gene and the risk of PV, 408 patients diagnosed with PV and 363 age and sex-matched healthy controls from a cohort of the Chinese majority Han population were recruited. Four single nucleotide polymorphisms (rs2232365, rs3761547, rs3761548 and rs3761549) of the FOXP3 gene were analyzed using the polymerase chain reaction and ligase detection reaction. The major allele of three single nucleotide polymorphisms (SNPs — rs2232365 A, rs3761547 A and rs3761549 C) were associated with an increased risk of PV in a clinical subgroup of female patients, who were less than 40 yrs of age, had a family history of the disease and did not have disease complications (p < 0.05 for all parameters). The haplotype was structured between rs3761547 and rs3761549. An increased risk of PV was observed in haplotype A/A-T/T (p = 0.0055; adjusted OR = 3.188; 95% CI = 0.4354-23.34) and A/G-C/C (p = 0.0082; adjusted OR = 1.288; 95% CI = 0.1529-10.85) between rs3761547 and rs3761549. A synergistic effect was found among the three SNPs. Subjects with the rs2232365AA- rs3761547 AG + GG genotype were more susceptible to PV (p = 0.0393; OR = 2.90; 95% CI = 1.05-7.97). No correlation was found between rs3761548 and the onset of PV. Therefore, the FOXP3 polymorphisms appear to contribute to the risk of psoriasis among the Chinese majority Han population. These findings may aid in our understanding of the pathogenesis of psoriasis

Leucine zippers improves protein splicing-mediated coagulation factor VIII gene delivery by dual-vector system / 药学学报

In our recent study by exploring an intein-based dual-vector to deliver a B-domain-deleted FVIII (BDD-FVIII) gene, it showed that covalently ligated intact BDD-FVIII molecules with a specific coagulant activity could be produced from expressed heavy and light chains by protein trans-splicing. Here, we assessed the hypothesis that the efficiency of trans-splicing may be increased by adding to the intein sequences a pair of leucine zippers that are known to bring about specific and strong protein binding. The intein-fused heavy and light chain genes were co-transferred into cultured COS-7 cells using a dual-vector system. After transient expression, the intracellular BDD-FVIII splicing was observed and the spliced BDD-FVIII and bioactivity secreted to culture media were quantitatively analyzed. An enhanced splicing of BDD-FVIII with decreased protein precursors from gene co-transfected cells was observed by Western blotting. The amount of spliced BDD-FVIII and bioactivity secreted to the culture media were 106 +/- 12 ng x mL(-1) and 0.89 +/- 0.11 U x mL(-1) analyzed by ELISA and Coatest method respectively, which was greater than leucine zipper free intein-fused heavy and light chain genes co-transfected cells (72 +/- 10 ng x mL(-1) and 0.62 +/- 0.07 U x mL(-1)). The activity of cellular mechanism-independent protein splicing was also improved, as showed by the increasing of spliced BDD-FVIII and bioactivity in culture media from combined cells separately transfected with heavy and light chain genes which was 36 +/- 11 ng x mL(-1) and 0.28 +/- 0.09 U x mL(-1). It demonstrated that the leucine zippers could be used to increase the efficiency of protein trans-splicing to improve the efficacy of a dual-vector mediated BDD-FVIII gene delivery by strengthening the interaction between the two intein-pieces fused to heavy and light chains. It provided evidence for further study in animal model using a dual-adeno-associated virus vector to deliver FVIII gene in vivo.

Glycosylation and L303e/F309S mutations improve intein-mediated splicing of the split coagulation factor VIII / 药学学报

We recently demonstrated that an intein-mediated protein splicing can be used to transfer B-domain-deleted FVIII (BDD-FVIII) gene by a dual-vector. In this study, we observed the effect of a variant heavy chain with six potential glycosylation sites of B domain and L303E/F309S mutations in its A1 domain, which were proven to be beneficial for FVIII secretion, on secretion of spliced BDD-FVIII. By transient co-transfection of cultured 293 cells with intein-fused variant heavy chain (DMN6HCIntN) and light chain (IntCLC) genes, the culture supernatant was analyzed quantitatively by ELISA for secreted spliced BDD-FVIII antigen and by a chromogenic assay for bioactivity. The data showed that the amount of spliced BDD-FVIII protein and coagulation activity in culture supernatant from DMN6HCIntN plus IntCLC co-transfected cells were up to (149 +/- 23) ng x mL(-1) and (1.12 +/- 0.14) u x mL(-1) respectively greater than that of intein-fused wild type heavy (HCIntN) and light chain (IntCLC) co-transfected cells [(99 +/- 14) ng x mL(-1) and (0.77 +/- 0.13) u x mL(-1)] indicating that the variant heavy chain is able to improve the secretion of spliced BDD-FVIII and activity. A cellular mechanism-independent BDD-FVIII splicing was also observed. It provided evidence for ongoing animal experiment using intein-mediated dual-AAV vector technology for delivery of the BDD-FVIII genes.

Protein trans-spliced chimeric human/porcine BDD-FVIII with augmented secretion / 药学学报

This study is to construct a chimeric human/porcine BDD-FVIII (BDD-hpFVIII) containing the substituted porcine A1 and A3 domains which proved to have a pro-secretory function. By exploring Ssp DnaB intein's protein trans-splicing a dual-vector was adopted to co-transfer the chimeric BDD-hpFVIII gene into cultured COS-7 cell to observe the intracellular BDD-hpFVIII splicing by Western blotting and secretion of spliced chimeric BDD-hp FVIII protein and bio-activity using ELISA and Coatest assay, respectively. The dada showed that an obvious protein band of spliced BDD-hpFVIII can be seen, and the amount of spliced BDD-hpFVIII protein and bio-activity in the supernatant were up to (340 +/- 64) ng x mL(-1) and (2.52 +/- 0.32) u x mL(-1) secreted by co-transfected cells which were significantly higher than that of dual-vector-mediated human BDD-FVIII gene co-transfection cells [(93 +/- 22) ng x mL(-1), (0.72 +/- 0.13) u x mL(-1)]. Furthermore, a spliced BDD-hpFVIII protein and activity can be detected in supernatant from combined cells separately transfected with intein-fused BDD-hpFVIII heavy and light chain genes indicating that intein-mediated BDD-hpFVIII splicing occurs independently of cellular mechanism. It provided evidence for enhancing FVIII secretion in the research of animal models using intein-based dual vector for the delivery of the BDD-hpFVIII gene.

Enhancing effect of porcine coagulation factor VIII A1 and A3 domains on secretion of post-translationally spliced human/porcine hybrid coagulation factor VIII / 生理学报

Low levels of coagulation factor VIII (fVIII) protein expression caused by its inefficient secretion and the over-sized fVIII gene affect the transgene-based gene therapy for hemophilia A adversely. Our previous study demonstrated that intein-mediated protein trans-splicing for delivery of the fVIII gene with a dual-vector system could improve secretion of post-translationally spliced fVIII by light chain in cis. In this study, a human/porcine hybrid fVIII (HP-fVIII) containing replaced A1 and A3 domains of porcine fVIII was investigated for secretion and activity of the spliced HP-fVIII after intein-based dual-vector delivery of the HP-fVIII gene. A pair of expression plasmids comprising intein-fused HP-fVIII heavy and light chains were constructed and transiently co-transfected into COS-7 cells. The spliced HP-fVIII and bio-activity in culture media were quantitatively analyzed by ELISA and Coatest method respectively. The intracellular splicing of HP-fVIII was detected by Western blotting. The results showed that in the culture supernatant of cells co-transfected with HP-fVIII, the amount and activity of spliced HP-fVIII were significantly higher than those of spliced hfVIII secreted from the cells co-transfected with human fVIII [(184+/-34 ng/mL) vs (48+/-12) ng/mL, P<0.01; (1.18+/-0.22) IU/mL vs (0.31+/-0.10) IU/mL, P<0.01], demonstrating the dramatically enhancing effect of porcine A1 and A3 domains on the secretion of intein-spliced HP-fVIII. The spliced HP-fVIII protein and its activity were also detected in the supernatant from combined cells separately transfected with intein-fused HP-fVIII heavy and light chain genes, indicating that the intein-mediated HP-fVIII splicing was independent of cellular mechanism and could occur outside the cell after the secretion of precursor proteins. Additionally, an intracellularly spliced HP-fVIII band was found with a molecular weight similar to human fVIII protein, confirming the HP-fVIII splicing. These results provided experimental basis for ongoing study using intein-based dual adeno-associated virus (AAV) vector to transfer HP-fVIII gene in animal models.

Post-translational ligation and function of dual-vector transferred split CFTR gene / 药学学报

The mutation of cystic fibrosis transmembrane conductance regulator (CFTR) gene leads to an autosomal recessive genetic disorder cystic fibrosis (CF). The gene therapy for CF using adeno-associated virus (AAV) vectors delivering CFTR gene is restricted by the contents limitation of AAV vectors. In this study the split CFTR genes severed at its regulatory domain were delivered by a dual-vector system with an intein-mediated protein trans-splicing as a technique to investigate the post-translational ligation of CFTR half proteins and its function as a chloride ion channel. A pair of eukaryotic expression vectors was constructed by breaking the human CFTR cDNA before Ser712 codon and fusing with Ssp DnaB intein coding sequences. After co-transfection into baby hamster kidney (BHK) cells followed by transient expression, patch clamps were carried out to record the chloride current of whole-cell and the activity of a single channel, and the ligation of two halves of CFTR was observed by Western blotting. The results showed that the intein-fused half genes co-transfected cells displayed a high whole cell chloride current and activity of a single channel indicating the functional recovery of chloride channel, and an intact CFTR protein band was figured out by CFTR-specific antibodies indicating that intein can efficiently ligate the separately expressed half CFTR proteins. The data demonstrated that protein splicing strategy could be used as a strategy in delivering CFTR gene by two vectors, encouraging our ongoing research program on dual AAV vector system based gene transfer in gene therapy for cystic fibrosis.

vWF improves secretion and activity of intein spliced BDD-FVIII / 药学学报

As synthesized by vascular endothelial cells and megakaryocytes, the von Willebrand factor (vWF) plays an important hemostatic role in the binding to and stabilizing blood coagulation factor VIII (FVIII) and preventing its enzymatic degradation. Our recent work demonstrated intein can efficiently ligate BDD-FVIII (B-domaim deleted FVIII) posttranslationally by protein trans-splicing after transfer of split BDD-FVIII gene by a dual-vector system. In this study we investigated the effect of vWF on secretion and activity of intein-ligated BDD-FVIII. We observed the levels of full-length BDD-FVIII antigen secreted into culture supernatant by ELISA and their activity by Coatest assay after transfection of cultured 293 cells with intein-fused BDD-FVIII heavy- and light-chain genes simultaneously with the vWF gene co-transfected. The data showed that the amount of full-length BDD-FVIII protein and their bioactivity in vWF gene co-transfected cell supernatant were 235 +/- 21 ng x mL(-1) and 1.98 +/- 0.2 u x mL(-1), respectively, greater than that of non-vWF co-transfected cell (110 +/- 18) ng x mL(-1) and 1.10 +/- 0.15 u x nL(-1)) or just BDD-FVIII gene transfected control cell (131 +/- 25 ng x mL(-1) and 1.22 +/- 0.18 u x mL(-1)) indicating the benefit of vWF gene co-transfection in the secretion and activity of intein-spliced BDD-FVIII protein. It provided evidence that vWF gene co-transfer may be useful to improve efficacy of gene therapy for hemophilia A in protein splicing-based split FVIII gene transfer.

Inteins in pathogenic fungi: a phylogenetic tool and perspectives for therapeutic applications

Inteins or "internal proteins" are coding sequences that are transcribed and translated with flanking sequences (exteins). After translation, the inteins are excised by an autocatalytic process and the host protein assumes its normal conformation and develops its expected function. These parasitic genetic elements have been found in important, conserved proteins in all three domains of life. Most of the eukaryotic inteins are present in the fungi kingdom and the PRP8 intein is one of the most widespread inteins, occurring in important pathogens such as Cryptococcus neoformans (varieties grubii and neoformans), Cryptococcus gattii, Histoplasma capsulatum and Paracoccidioides brasiliensis. The knowledge of conserved and non-conserved domains in inteins have opened up new opportunities for the study of population variability in pathogenic fungi, including their phylogenetic relationships and recognition or diagnoses of species. Furthermore, inteins in pathogenic fungi should also be considered a promising therapeutic drug target, since once the autocatalytic splicing is inhibited, the host protein, which is typically vital, will not be able to perform its normal function and the fungal cell will not survive or reproduce.

Intein-mediated F309SfVIII ligation with enhanced secretion of its heavy chain / 生理学报

Coagulation factor VIII (fVIII) is a secretion protein and plays a crucial role in the coagulation cascade. Hemophilia A resulted from deficiency of fVIII is the most common X-linked recessive bleeding disorder. Gene therapy is recognized as an attractive strategy for the eventual cure of this disease. However, the gene therapy is hampered by the big size of fVIII gene when using the most promising gene vectors, adeno-associated virus (AAV) vectors. In this study we explored the intein-mediated protein trans-splicing to deliver a Phe(309)-->Ser mutant full-length fVIII (F309SfVIII) gene by using a dual-vector system. An intein is a protein sequence embedded within a precursor protein and can excise itself through protein splicing. The F309SfVIII is proven to be beneficial to its secretion. The F309SfVIII gene was broken into heavy and light chains before Ser(1239) in B domain and fused with the coding sequences of Ssp DnaB intein respectively to construct a pair of plasmid vectors by inserting them into the pcDNA3.1 vectors. Forty-eight hours after co- or separate transfection of 293 cells, the co-transfected cell lysate showed an obvious ligated F309SfVIII protein band by Western blot with a polyclonal antibody against fVIII. The amounts of secreted F309SfVIII protein in culture supernatants and their bioactivities were (71+/-9) ng/mL and (0.38+/-0.09) IU/mL determined by ELISA and Coatest assay respectively. The supernatant from combined cells with separate transfections also displayed lower levels of F309SfVIII antigen and fVIII activity [(25+/-6) ng/mL and (0.12+/-0.05) IU/mL], indicating the F309SfVIII could be formed by splicing both before and after secretion. The content of F309SfVIII heavy chain protein from co-transfected cell supernatant was higher than that of intein-fused heavy chain transfection alone [(135+/-10) ng/mL vs (37+/-7) ng/mL, P<0.01)]. These data demonstrated that intein could be used as a technical strategy in a dual-vector system delivering F309SfVIII gene with improved secretion of fVIII providing an alternative approach to circumvent the packaging limitation of AAV for F309SfVIII gene transfer, which encourages our continuing study in hemophilia A gene therapy in vivo.

Ssp DnaB intein-mediated ligation of heavy and light chains of coagulation factor VIII in Escherichia coli / 生物工程学报

We studied the ligation of coagulation factor VIII heavy and light chains in Escherichia coli by utilizing the intein-mediated protein trans-splicing. A B-domain deleted factor VIII (BDD-FVIII) gene was broken into two halves of heavy and light chains before Ser1657 which meets the splicing required conserved residue and then fused to 106 and 48 amino acid-containing N-part termed Int-N and C-part termed Int-C coding sequences of split mini Ssp DnaB intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pBV220. Through induction for expression of recombinant protein it displayed an obvious protein band as predicted size of BDD-FVIII protein on SDS-PAGE gel. Western blotting using factor VIII specific antibodies confirmed that this protein band is BDD-FVIII produced by protein trans-splicing. It demonstrated that the heavy and light chains of BDD-FVIII can be efficiently ligated with the Ssp DnaB intein-mediated protein trans-splicing. These results provided evidence for encouraging our ongoing investigation with intein as a means in dual AAV vectors carrying the factor VIII gene to overcome the packaging size limitation of a single AAV vector in hemophilia A gene therapy.

Molecular cloning of chitinase 33 (chit33) gene from Trichoderma atroviride / Clonagem molecular do gene quitinase 33 (chit33) em Trichoderma atroviride

In this study Trichoderma atroviride was selected as over producer of chitinase enzyme among 30 different isolates of Trichoderma sp. on the basis of chitinase specific activity. From this isolate the genomic and cDNA clones encoding chit33 have been isolated and sequenced. Comparison of genomic and cDNA sequences for defining gene structure indicates that this gene contains three short introns and also an open reading frame coding for a protein of 321 amino acids. The deduced amino acid sequence includes a 19 aa putative signal peptide. Homology between this sequence and other reported Trichoderma Chit33 proteins are discussed. The coding sequence of chit33 gene was cloned in pEt26b(+) expression vector and expressed in E. coli.

Neste estudo Trichoderma atroviride foi escolhido como superprodutor da enzima quitinase dentre 30 isolados de Trichoderma sp. com base na atividade específica de quitinase. Clones de cDNA e genômico codificando chit33 foram obtidos deste isolado e seqüenciados. A comparação das seqüências genômica e de cDNA para definir a estrutura do gene indicou que este contém três pequenos introns e uma fase aberta de leitura codificando uma proteína de 321 aminoácidos. A seqüência de aminoácidos deduzida inclui um possível peptídio sinal de 19 aminoácidos. Homologia entre esta seqüência e outras proteínas Chit33 descritas de Trichoderma é discutida. A seqüência codificadora do gene chit33 foi clonada no vetor de expressão pET26b(+) e expressa em E. coli.

Construction and evaluation of DnaB split intein high expression vector and a six amino acids cyclic peptide library / 生物工程学报

A library with potential to produce six amino acids cyclic peptides was prepared using pET-28a as the starting plasmid. pVmut was used to amplify the Int(C)-dnaB-N-Int(N) fragment that was inserted into pET28a to give pEV. On pEV, DnaB split intein was expressed under the strong T7 promoter. Analyses of Escherichia coli transformed with pEV showed that DnaB split intein was produced in large quantity and the fusion protein self-spliced efficiently to produce cyclized DnaB-N. A synthesized 115 bp fragment mixture encoding 5 random amino acids was inserted into pEV to generate pEV-IS. The ligation mixture was transformed into E. coli. A library of 10(3) clones was obtained, 20 randomly picked clones were sequenced. All of them contain different sequences. Nine clones were chosen for further analysis. Split-intein-ISs were expressed in large quantity, and 90% of them self-spliced under 16 degrees C in 20 hours. After induction at 30 degrees C for 3 hours, the expressed DnaB split intein was purified using His-column, and then a molecular weight of target cyclic peptide was detected by MALDI-TOF-MS.

Preparation and identification of recombinant maxadilan / 生物工程学报

To produce recombinant Maxadilan using gene engineering technology, the gene of recombinant Maxadilan which expressed in protocaryon were designed and synthesized according to the amino acid sequences of Maxadilan. The recombinant plasmid pKYB-MAX was constructed and transformed into host bacteria Escherichia coli strain ER2566. After the MAX-intein-CBD fusion protein was purified by chintin-affinity chromatography, the self-cleavage activity of the intein was induced by beta-mercaptoethanol and the recombinant Maxadilan was released from the chitin-bound intein tag. The molecular weight of peptides was determined by the laser flight mass spectrometry and the results was conformity with the theoretical value. The biological activity analysis showed that recombinant Maxadilan significantly enhanced the concentration of serum glucose.

Soluble expression, purification and characterization of Bm K IT in Escherichia coli by intein-mediated system / 生物工程学报

To produce recombinant Buthus martensii Karsch insect toxin (BmK IT), BmK IT cDNA which fused a hexahistidine sequence at the C-terminus by PCR was inserted into pTWIN1 expression vector fused in frame with an upstream Ssp DnaB intein gene. The expression plasmid was transformed into E. coli BL21 (DE3) strain and protein expression was induced by IPTG. The CBD-Intein-BmK IT(his6) fusion protein was purified from cell lysates using Ni-NTA resin affinity chromatography. The intein was removed from fusion protein by on-column intein-mediated cleavage. BmK IT(his6) was purified through Superdex 75 gel chromatography to more than 95% homogeneity. The purified protein has both correct secondary structure and insecticidal activity.

Use of Ssp dnaB mini-intein as a fusion partner for preparation of recombinant human brain natriuretic peptide / 生物工程学报

Human brain natriuretic peptide (hBNP) was used clinically for the treatment of acute decompensated congestive heart failure. In this paper, hBNP was expressed as a fusion protein with a histidine tag and Ssp dnaB mini-intein which was capable of self-cleavage. After affinity chromatography with Ni-Sepharose and renaturation, the fusion protein was enriched with CM-cellulose. Ssp dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH and temperature in the CM-cellulose column, which let to the release of hBNP from the fusion protein and the separation of hBNP from His-DnaB. The hBNP sample was further purified by C4 reverse phase HPLC, and 2.8mg of the peptide with homogeneity of 97% was obtained from one liter of culture medium. The biological activity was assayed in vitro, which indicated that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC50 of 1.94 x 10(-6) mg/mL.

Effect of the Heat Shock Protein 70 on the Adjuvanticity Induced by a Bacterial Flagellin of Vibrio ulnificus

Recently we have shown that a bacterial flagellin, Vibrio vulnifiucs FlaB (Vv-FlaB), has a strong adjuvant activity to induce protective immune response. In order to investigate the adjuvanticity of Vv-FlaB, we prepared highly purified recombinant protein by using an intein fusion protein purification system. However, in the process of the purification, we unexpectedly encountered a contamination with a 70 kDa protein. We proved the 70 kDa protein as the heat shock protein 70 (HSP70) by Western blotting. Unfortunately, it was reported that the HSP70 has a strong adjuvanticity. In this study we investigated the role of contaminating HSP70 on the Vv-FlaB-mediated adjuvanticity. We separated Vv-FlaB and HSP70 by using a high performance protein purification chromatography and compared adjuvant activities of Vv-FlaB, HSP70 and Vv-FlaB/HSP70 mixture. Using an intranasal immunization mouse model, we observed that co-administration of the flagellin with tetanus toxoid (TT) induced significantly enhanced TT-specific antibody (Ig) responses. However contaminating doses of HSP70 did not affect the adjuvanticity of Vv-FlaB and furthermore HSP70 alone did not enhance TT-specific Ig response and protective immunity against lethal challenge with tetanus toxin. These results show that the HSP70 contaminating Vv-FlaB preparations did not affect the adjuvanticity of Vv-FlaB.