His-tagged Fusion Protein Research Articles

English

Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease that has devastated the swine industry worldwide. Vaccination with live attenuated vaccine or inactivated vaccine is the main treatment to control PRRS. However, the disadvantages such as virulence resumption of the attenuated vaccine and low immunogenicity of the inactivated vaccine call for a more efficient and safer genetically engineered vaccine. In this study, the structural protein GP5 of the PRRS virus (PRRSV), one of the major protective antigens which stimulates a protective immune response was selected to develop a genetically engineered subunit vaccine. In order to promote the immune reaction of the host to GP5, heat shock protein 70 (Hsp70) was selected as immuno-adjuvant to enhance PRRSV GP5 immunogenicity. The Hsp70 gene was amplified by PCR from attenuated Mycobacterium bovis, and the PRRSV GP5 gene was amplified by RT-PCR from the total RNA of PRRSV SCQ strain which was isolated, identified and maintained by the Animal Biotechnological Center, Sichuan Agricultural University, China. The fusion expressing plasmid pET32-GP5-Hsp70 was constructed and expressed in Escherichia coli BL21. Ni2+-chelating resin was used to purify the His-tagged fusion protein expressed under optimized expressing conditions. The rabbit anti-GP5-Hsp70 fusion protein antibody was made, and Western blot assay verified the successful expression of the fusion protein, making it possible for further investigation whether Hsp70 could improve the immunogenicity of the PRRSV GP5 subunit vaccine, or evaluating the immunogenicity of the GP5-Hsp70 subunit vaccine. Key words: Porcine reproductive and respiratory syndrome virus (PRRSV) GP5 gene, Mycobacterium bovis Hsp70 gene, cloning, prokaryotic expression, identification.

Expression, purification, and enzymatic characterization of Bombyx mori nucleopolyhedrovirus DNA polymerase

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms. BmNPV DNA polymerase (Bm-DNAPOL), encoded by the ORF53 gene, plays a central role in viral DNA replication. In this work, a His-tagged Bm-DNAPOL fusion protein, constructed using a novel MultiBac expression system, was overexpressed in Sf-9 insect cells, purified to near homogeneity on Ni-NTA agarose beads and further purified by ion-exchange chromatography. About 0.4 mg of enzyme was obtained from about 1 × 10(9) infected Sf-9 cells in suspension culture. Characterization of the highly purified enzyme indicated that Bm-DNAPOL is a monomer with an apparent molecular mass of approximately 110,000 Da. It possessed a specific activity of 15,126.3 U/mg under optimal in vitro reaction conditions and behaved in the manner of a proliferating cell nuclear antigen (PCNA)-independent DNA polymerase on both poly(dA)/oligo(dT) primer/template and singly premiered M13 DNA. BmNPV viral replication may be independent of replication factor C and a PCNA complex, while single-stranded DNA binding protein might play an important role in BmNPV DNA replication. These findings will be significant in studies on BmNPV-based disease in silkworms and for using silkworms as a bioreactor for the production of biomolecules of commercial importance.

Monoclonal Antibodies Against Tyrosyl-tRNA Synthetase and Its Isolated Cytokine-Like Domain

Tyrosyl-tRNA synthetase (TyrRS) is one of the key enzymes of protein biosynthesis. In addition to its basic role, this enzyme reveals some important non-canonical functions. Under apoptotic conditions, the full-length enzyme splits into two fragments having distinct cytokine activities, thereby linking protein synthesis to cytokine signaling pathways. The NH2-terminal catalytic fragment, known as miniTyrRS, binds strongly to the CXC-chemokine receptor CXCR1 and, like interleukin 8, functions as a chemoattractant for polymorphonuclear leukocytes. On the other hand, an extra COOH-terminal domain of human TyrRS has cytokine activities like those of a mature human endothelial monocyte-activating polypeptide II (EMAP II). Moreover, the etiology of specific diseases (cancer, neuronal pathologies, autoimmune disorders, and disrupted metabolic conditions) is connected to specific aminoacyl-tRNA synthetases. Here we report the generation and characterization of monoclonal antibodies specific to N- and C-terminal domains of TyrRS. Recombinant TyrRS and its N- and C-terminal domains were expressed as His-tag fusion proteins in bacteria. Affinity purified proteins have been used as antigens for immunization and hybridoma cell screening. Monoclonal antibodies specific to catalytic N-terminal module and C-terminal EMAP II-like domain of TyrRS may be useful as tools in various aspects of TyrRS function and cellular localization.

A Novel Hyaluronidase from Brown Spider (Loxosceles intermedia) Venom (Dietrich's Hyaluronidase): From Cloning to Functional Characterization

Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. The venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide and a mature protein. Amino acid alignment revealed a structural relationship with members of hyaluronidase family, such as scorpion and snake species. Recombinant hyaluronidase was expressed as N-terminal His-tag fusion protein (∼45 kDa) in inclusion bodies and activity was achieved using refolding. Immunoblot analysis showed that antibodies that recognize the recombinant protein cross-reacted with hyaluronidase from whole venom as well as an anti-venom serum reacted with recombinant protein. Recombinant hyaluronidase was able to degrade purified hyaluronic acid (HA) and chondroitin sulfate (CS), while dermatan sulfate (DS) and heparan sulfate (HS) were not affected. Zymograph experiments resulted in ∼45 kDa lytic zones in hyaluronic acid (HA) and chondroitin sulfate (CS) substrates. Through in vivo experiments of dermonecrosis using rabbit skin, the recombinant hyaluronidase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin from L. intermedia venom (LiRecDT1). These data support the hypothesis that hyaluronidase is a “spreading factor”. Recombinant hyaluronidase provides a useful tool for biotechnological ends. We propose the name Dietrich's Hyaluronidase for this enzyme, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglycans.

High yield expression of duck hepatitis A virus VP1 protein in Escherichia coli, and production and characterization of polyclonal antibody

VP1 protein, the capsid protein of duck hepatitis A virus (DHAV), contains critical epitopes for inducing a protective immune response. Due to its low-level expression in Escherichia coli (E. coli), the function of this protein is poorly characterized. In this study, a codon-optimized VP1 gene was chemically synthesized in terms of the codon usage bias in E. coli and subcloned into pET32a (+) to increase its expression. The recombinant VP1 fusion protein was purified from inclusion body by Ni2+ affinity chromatography His-Bind Resin and used to raise the rabbit anti-DHAV-VP1 polyclonal antibody. The expression of the codon-optimized VP1 gene in E. coli was significantly increased when compared to the wild-type VP1 gene, having an at least 17-fold increase. Western blot analysis showed that the recombinant protein was recognized by the rabbit anti-DHAV polyclonal antibody. Western blot also demonstrated that the rabbit anti-DHAV-VP1 polyclonal antibody could recognize the purified VP1 fusion protein specifically, and in the indirect immunofluorescent assays (IFA), the antibody was able to probe the VP1 protein in DHAV-1 infected cells. In conclusion, codon optimization increased dramatically DHAV VP1 expression in E. coli and the His-tagged VP1 fusion protein showed good antigenicity and immunogenicity.

Coupled solid phase extraction and microparticle-based stability and purity-indicating immunosensor for the determination of recombinant human myelin basic protein in transgenic milk

An optical immunosensor was developed and validated on the surface of microparticles for the determination of a biopharmaceutical protein. The recombinant human myelin basic protein (rhMBP) was produced in milk of transgenic cows as a His-tagged fusion protein. Previous work indicated exclusive association of rhMBP with milk casein micelles that hindered direct determination of the protein in milk. In this work, a solid phase extraction using a cation exchange matrix was developed in order to liberate rhMBP from casein micelles. A sandwich-type immunoassay was then used for in-process monitoring of the full-length protein in the presence of major milk proteins. The assay was successfully employed for detection of ultra-traces of rhMBP (LOD=6.04ngmL−1≈0.3nmolL−1) and for quantitative determination over a wide concentration range (10.00–10,000.00ngmL−1). The assay was able also to detect the rhMBP in the presence of its human counterpart that lacks the His-tag. The high sensitivity along with the ability of the assay to determine the full length protein enabled monitoring of the stability of rhMBP. The testing protocol is particularly useful for intrinsically unstructured proteins that are extremely sensitive to proteolysis and lack a traceable enzymatic activity. This immunosensor provides a specific, ultrasensitive high throughput tool for in-process monitoring in biopharmaceutical industry.

Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli

Thionins are antimicrobial plant peptides produced as preproproteins consisting of a signal peptide, the thionin domain, and a so-called acidic domain. Only thionin itself has been isolated from plants. To study the processing of the precursor, it has to be produced in a heterologous system. Since both domains contain several cysteines and, due to the known antimicrobial activity of the thionin, we tested the expression of all four Arabidopsis proproteins as fusion proteins. Periplasmic expression as fusion with maltose binding protein was not successful but cytoplasmic expression as His-tagged TRX fusion proteins with a TEV recognition sequence resulted in proteins of correct size. Use of the SHuffle strain C3030 further improved the expression. Fusion proteins inhibited growth of Escherichia coli. They could be cleaved by TEV protease, releasing authentic proproteins without any additional amino acid at the N-terminus.Electronic supplementary materialThe online version of this article (doi:10.1007/s10529-013-1180-z) contains supplementary material, which is available to authorized users.

Production and characterization of mouse monoclonal antibodies against lysis protein E of phiX174

Bacterial ghosts offer a new avenue for the study of inactivated vaccines. However, for many years the mechanism of genetic inactivation was controversial. To obtain mouse monoclonal antibodies (mAbs) against protein E will allow the observation of its location and dynamic expression to expand understanding of the lysis mechanism. In this study, a His-tagged ΔE fusion protein expressed in bacteria was used as the immunogen, and mAbs targeting protein E were produced by the hybridoma technique and selected by enzyme-linked immunosorbent assay using GST-E and GST-ΔE as coating proteins. Purified mAbs from mouse ascites were screened against a phage-displayed random dodecapeptide library (Ph.D.-12). After three rounds of biopanning, 30 phage clones were randomly selected and sequenced, and their amino acids were deduced. One epitope showed a good match with protein E at 57–63aa (KPLN--R) and the synthetic decapeptide Epep (VRLKPLNCSR) strongly inhibited combination of E-A5 with E fusion proteins. Immunofluorescence microscopy indicated specific immunoreactivity of E-A5 with Escherichia coli-expressed native protein E. The novel mAbs may be of great potential value in analysis of the function and lysis pathway of protein E and other relative phages and in evaluation of E as potential marker of bacterial ghosts.

Trehalose phosphate synthases OtsA1 and OtsA2 ofRhodococcus opacus1CP

Rhodococcus opacus 1CP produces trehalose dinocardiomycolates during growth on long-chained n-alkanes. Trehalose and trehalose-6-phosphate, which are synthesized via the OtsAB pathway, are probable intermediates in the biosynthesis of these biosurfactants. By molecular genetic screening for trehalose-6-phosphate synthases (TPSs and OtsAs), two chromosomal fragments of strain 1CP were obtained. Each contained an ORF whose amino acid sequence showed high similarity to TPSs. To prove the activity of the otsA1 and otsA2 gene product and to detect catalytic differences, both were expressed as His-tagged fusion proteins. Enzyme kinetics of the enriched proteins using several potential glucosyl acceptors showed an exclusive preference for glucose-6-phosphate. In contrast, both enzymes were shown to differ significantly from each other in their activity with different glucosyl nucleotides as glucosyl donors. OtsA1-His10 showed highest activity with ADP-glucose and UDP-glucose, whereas OtsA2-His10 preferred UDP-glucose. In addition, the wild-type OtsA activity of R.opacus 1CP was investigated and compared with recombinant enzymes. Results indicate that OstA2 mainly contributes to the trehalose pool of strain 1CP. OtsA1 seems to be involved in the overproduction of trehalose lipids. For the first time, a physiological role of two different OtsAs obtained of a single Rhodococcus strain was presumed.

A New Strategy for Intracellular Delivery of Enzyme Using Mesoporous Silica Nanoparticles: Superoxide Dismutase

We developed mesoporous silica nanoparticle (MSN) as a multifunctional vehicle for enzyme delivery. Enhanced transmembrane delivery of a superoxide dismutase (SOD) enzyme embedded in MSN was demonstrated. Conjugation of the cell-penetrating peptide derived from the human immunodeficiency virus 1 (HIV) transactivator protein (TAT) to mesoporous silica nanoparticle is shown to be an effective way to enhance transmembrane delivery of nanoparticles for intracellular and molecular therapy. Cu,Zn-superoxide dismutase (SOD) is a key antioxidant enzyme that detoxifies intracellular reactive oxygen species, ROS, thereby protecting cells from oxidative damage. In this study, we fused a human Cu,Zn-SOD gene with TAT in a bacterial expression vector to produce a genetic in-frame His-tagged TAT-SOD fusion protein. The His-tagged TAT-SOD fusion protein was expressed in E. coli using IPTG induction and purified using FMSN-Ni-NTA. The purified TAT-SOD was conjugated to FITC-MSN forming FMSN-TAT-SOD. The effectiveness of FMSN-TAT-SOD as an agent against ROS was investigated, which included the level of ROS and apoptosis after free radicals induction and functional recovery after ROS damage. Confocal microscopy on live unfixed cells and flow cytometry analysis showed characteristic nonendosomal distribution of FMSN-TAT-SOD. Results suggested that FMSN-TAT-SOD may provide a strategy for the therapeutic delivery of antioxidant enzymes that protect cells from ROS damage.

Cloning, Expression and Purification of L. Donovani Specific Antigen for Serodiagnosis of Visceral Leishmaniasis

Rapid diagnostic test using rk39 antigen is widely used for visceral leishmaniasis. However it detects anti-rk39 antibodies in 20-32% of endemic healthy individuals. In search for a better biomarker of infection, we identified a protein of molecular weight 70 kDa (BHUP1), specifically recognized by sera of visceral leishmaniasis (VL) patients. The protein was cloned as His-tagged fusion protein and purified. We evaluated the sensitivity and specificity of this protein in an enzyme linked immunosorbant assay (ELISA) format in comparison to the rk39 antigen using sera collected from various groups of individuals. The sensitivity of rBHUP1 was 96.5% compared to 98.8% with rk39. For healthy controls from non endemic and endemic regions, the specificity of rBHUP1 was 100% and 95.6% compared to 100% and 84.9% for rk39, respectively. For other infectious diseases such as malaria, tuberculosis, viral fever, etc., specificity of rBHUP1 was as low as 74.5% when compared to 94% of rk39. At six month and one year follow-up, 74% and 22.5% patients tested positive with rBHUP1, respectively, compared to 97% and 77.4% with rk39 antigen. Though the high sensitivity and specificity of rBHUP1 antigen for VL and healthy controls would have made it a good diagnostic biomarkers, however, its non-specific reaction with other infectious diseases limit its utility.

Prokaryotic expression of a truncated form of bovine herpesvirus 1 glycoprotein E (gE) and its use in an ELISA for gE antibodies

This article describes the expression of a truncated form of bovine herpesvirus 1 (BoHV-1) glycoprotein E (gE) for use as immunodiagnostic reagent. A 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids) of BoHV-1 gE - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector. A soluble protein of approximately 25 kDa purified from lysates of transformed E. coli was recognized in Western blot (WB) by anti-6xHis-tag and anti-BoHV-1 gE monoclonal antibodies. In addition, the recombinant protein was specifically recognized in WB by antibodies present in the sera of cattle seropositive to BoHV-1 and BoHV-5. An indirect ELISA using the expressed protein as coating antigen performed comparably to a commercial anti-gE ELISA and was able to differentiate serologically calves vaccinated with a gE-deleted BoHV-5 strain from calves infected with BoHV-1. Thus, the truncated gE may be useful for serological tests designed to differentiate BoHV-1/BoHV-5 infected animals from those vaccinated with gE-negative marker vaccines.

Secretory expression of thermostable alkaline protease from <i>Bacillus stearothermophilus</i> FI by using native signal peptide and <b>α</b>-factor secretion signal in <i>Pichia pastoris</i>

The thermostable alkaline protease from Bacillus stearothermophilus F1 has high potential for industrial applications, and attempt to produce the enzyme in yeast for higher yield was undertaken. Secretory expression of F1 protease through yeast system could improve enzyme's capability, thus simplifying the purification steps. Mature and full genes of F1 protease were cloned into Pichia pastoris expression vectors (pGAPZαB and pPICZαB) and transformed into P. pastoris strains (GS115 and SMD1168H) via electroporation method. Recombinant F1 protease under regulation constitutive GAP promoter revealed that the highest expression was achieved after 72 h cultivation. While inducible AOX promoter showed that 0.5% (v/v) methanol was the best to induce expression. It was proven that constitutive expression strategy was better than inducible system. The α-secretion signal from the plasmid demonstrated higher secretory expression level of F1 protease as compared to native Open Reading Frame (ORF) in GS115 strain (GE6GS). Production medium YPTD was found to be the best for F1 protease expression with the highest yield of 4.13 U/mL. The protein was expressed as His-tagged fusion protein with a size about 34 kDa.

Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli

Chimeric peptide MVF-EGFR237–267, comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His6-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His6-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×104 titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides.

Molecular Cloning, Biochemical Characterization, and Differential Expression of an Acetyl-CoA C-Acetyltransferase Gene (AACT) of Brahmi (Bacopa monniera)

Bacopa monniera (Brahmi) is an important Indian medicinal herb found in wet damp and marshy places. It produces medicinally important compounds known as bacosides along with alkaloids like brahmine and herpestine. Bacosides are triterpenoid saponins and their biosynthesis takes place via the isoprenoid pathway starting with acetyl-CoA. Acetyl-CoA C-acetyltransferase (AACT; EC 2.3.1.9), also known as acetoacetyl-CoA thiolase (Thiolase II), catalyzes the condensation of two acetyl-CoA to form 4-C compound acetoacetyl-CoA. Acetoacetyl-CoA is an important starting molecule for biosynthesis of various metabolites. Here, we report the cDNA cloning and characterization of acetyl-CoA C-acetyltransferase gene from B. monniera. The full-length gene was isolated using a RACE PCR protocol. The cDNA encoding AACT was designated as BmAACT (FJ947159) revealed an ORF of 1,218 bp and 405 amino acids, and shares 80 % similarity with other plant AACTs. Phylogenetic analysis showed that BmAACT is related closely to other dicot plants AACTs. The BmAACT gene was over-expressed in Escherichia coli as a 6X His-tag fusion protein and purified to homogeneity by Ni-NTA and gel filtration chromatography. Activity of recombinant protein was confirmed by thiolytic cleavage of acetoacetyl-CoA in the presence of 5 mM Mg2+, showing K m and V max of 20.67 μM and 96.21 μmol/min, respectively, with high catalytic efficiency (k cat = 2.30 × 105 min−1). Quantitative real-time PCR analysis showed that the expression of BmAACT is tissue-specific, and accumulation of transcripts is greater in roots and petals, followed by sepals, stem, leaf and pedicel.

Plasmodium translationally repressed gene products are essential for parasite development and malaria transmission

Materials and methods RIP-Chip and RT-PCR of immunoprecipitated DOZI and CITH mRNPs from the rodent malaria parasite P. berghei were used to identify molecules with clear surface targeting signals that are associated with P body-like mRNPs. Targeted gene deletion and GFP-tagging was used to identify the function, sub-cellular localisation and expression patterns of these proteins. To study the development of knock-out parasite lines, Anopheles stephensi mosquitoes were allowed to feed on infected mice and infection was quantified at the oocyst and sporozoite stages. In silico analysis identified highly immunogenic peptides in each protein; they were concatenated as codon-optimised, chimerical His6-tagged fusion proteins for heterologous expression to be used in transmission blocking assays. Results 22 mRNAs encoding for surface proteins were shown to be associated with both DOZI- and CITH-defined mRNPs. These include pb25, pb28 ,t he entirepb-fam-5 family, as well as 12 uncharacterised gene products with orthologs in P. falciparum and P. vivax; the latter were targeted for gene deletion. Several of the knock-out mutants show a clear impairment in mosquito stage development, either at the oocyst or sporozoite levels. Some of these completely fail to transmit to naive mice. Full length and concatenated versions (superantigens) of these proteins were expressed in Escherichia coli BL21. Superantigen expression was achieved more easily and in higher yields than full-length proteins. Conclusions Here we identify novel P. berghei translationally repressed mRNAs that encode for mosquito stage surface proteins and are important for parasite development within its vector. Parasites lacking some of these proteins fail to transmit to naive hosts, and are therefore attractive targets for novel transmission blocking vaccines. Heterologous expression of Plasmodium protein is frequently a challenging task due to their disordered nature and the A/T richness of the genome. We used codon optimisation to compensate for A/T rich genes and developed a superantigen strategy to combine the most immunogenic regions of several proteins while avoiding their hydrophobic domains.

High Inorganic Triphosphatase Activities in Bacteria and Mammalian Cells: Identification of the Enzymes Involved

BackgroundWe recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities.Methodology/Principal FindingsRecombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPPi) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPPi but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility.Conclusions and General SignificanceWe show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPPi in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPPi, which could be cytotoxic because of its high affinity for Ca2+, thereby interfering with Ca2+ signaling.

Kinetic Analysis, Expression Pattern, and Production of a Recombinant Fungal Protease Inhibitor of Tasar Silkworm Antheraea mylitta

Antheraea mylitta, a tasar silk-producing insect of Saturniidae family, expresses a fungal protease inhibitor named as A. mylitta fungal protease inhibitor-1 (AmFPI-1). AmFPI-1 inhibits alkaline protease of Aspergillus oryzae but its mechanism of action is not known. To understand the mode of inhibition of AmFPI-1 against the fungal protease, it was purified from the hemolymph of A. mylitta larvae and inhibitory activity against A. oryzae protease was studied. Kinetic analysis of purified AmFPI-1 on alkaline protease of A. oryzae showed that AmFPI-1 acts as a canonical-type competitive inhibitor with equilibrium dissociation constant (K ( i )) of 60nM. Expression of AmFPI-1 in different body tissues of fifth instar A. mylitta larvae was determined by real-time PCR, and the highest expression was observed in fat body followed by integument, silk gland, and gut, indicating that AmFPI-1 has pleiotropic functions including protection from invading fungi. The cDNA of AmFPI-1 was expressed in Escherichia coli, and recombinant His-tagged fusion protein was purified by Ni-NTA chromatography. Recombinant AmFPI-1 showed inhibitory activity against A. oryzae protease and suggested its use in various biological applications to prevent proteolysis.

A Novel Nuclear Trafficking Module Regulates the Nucleocytoplasmic Localization of the Rabies Virus Interferon Antagonist, P Protein

Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1-P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3-P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection.

Expression of recombinant human cathepsin K is enhanced by codon optimization

A synthetic codon-optimized gene encoding human procathepsin K has been cloned in Escherichia coli using pET28a+ vector. The recombinant His-tagged fusion protein was expressed as inclusion body, solubilized in urea and purified by metal affinity chromatography. The purified protein was refolded by dilution technique, concentrated and finally purified by gel-filtration chromatography. The expressed protein was confirmed by Western blot analysis with human cathepsin K specific antibody. We have obtained 140mg purified and refolded protein from 1L bacterial culture which is the highest (nearly three times higher) yield reported so far for a recombinant human procathepsin K. The protease could be autocatalytically activated to mature protease at lower pH in presence of cysteine protease specific activators. The recombinant protease showed gelatinolytic and collagenolytic activities as well as activity against synthetic substrate Z-FR-AMC with a Km value of 5±2.7μM and the proteolytic activity of the enzyme could be blocked by cysteine protease inhibitors E-64, leupeptin and MMTS.