Self-assembling nanoparticles (saNP) and nanofibers were found in the recombinant coronavirus SARS-CoV-2 S1, S2, RBD and N proteins purified by affinity chromatography using Ni Sepharose. Scanning electron (SEM), atomic force (AFM) microscopy on mica or graphite surface and in liquid as well as dynamic light scattering (DLS) revealed nanostructures of various sizes. AFM in liquid cell without drying on the surface showed mean height of S1 saNP 80.03 nm, polydispersity index (PDI) 0.006; for S2 saNP mean height 93.32 nm, PDI = 0.008; for N saNP mean height 16.71 nm, PDI = 0.99; for RBD saNP mean height 16.25 nm, PDI = 0.55. Ratios between the height and radius of each saNP in the range 0.1-0.5 suggested solid protein NP but not vesicles with internal empty spaces. The solid but not empty structures of the protein saNP were also confirmed by STEM after treatment of saNP with the standard contrasting agent uranyl acetate. The saNP remained stable after multiple freeze-thaw cycles in water and hyperosmotic solutions for 2 years at -20 °C. Receptor-mediated penetration of the SARS-CoV-2 S1 and RBD saNP in the African green mokey kidney Vero cells with the specific receptors for β-coronavirus reproduction was more efficient compared to unspecific endocytosis into MDCK cells without the specific receptors. Amyloid-like structures were revealed in the SARS-CoV-2 S1, S2, RBD and N saNP by means of their interaction with Thioflavin T and Congo Red dyes. Taken together, spontaneous formation of the amyloid-like self-assembling nanostructures due to the internal affinity of the SARS-CoV-2 virion proteins might induce proteinopathy in patients, including conformational neurodegenerative diseases, change stability of vaccines and diagnostic systems.
The broad species tropism of Nipah virus (NiV) coupled with its high pathogenicity demand a rapid search for a new biomarker candidate for diagnosis. The matrix (M) protein was expressed in Escherichia coli and purified using a Ni-NTA affinity column chromatography and sucrose density gradient centrifugation. The recombinant M protein with the molecular mass (Mr) of about 43 kDa was detected by anti-NiV serum and anti-myc antibody. About 50% of the M protein was found to be soluble and localized in cytoplasm when the cells were grown at 30 degrees C. Electron microscopic analysis showed that the purified M protein assembled into spherical particles of different sizes with diameters ranging from 20 to 50 nm. The purified M protein showed significant reactivity with the swine sera collected during the NiV outbreak, demonstrating its potential as a diagnostic reagent.
The nucleocapsid (N) protein of Nipah virus (NiV) can be produced in three Escherichia coli strains [TOP10, BL21(DE3) and SG935] under the control of trc promoter. However, most of the product existed in the form of insoluble inclusion bodies. There was no improvement in the solubility of the product when this protein was placed under the control of T7 promoter. However, the solubility of the N protein was significantly improved by lowering the growth temperature of E. coli BL21(DE3) cell cultures. Solubility analysis of N- and C-terminally deleted mutants revealed that the full-length N protein has the highest solubility. The soluble N protein could be purified efficiently by sucrose gradient centrifugation and nickel affinity chromatography. Electron microscopic analysis of the purified product revealed that the N protein assembled into herringbone-like particles of different lengths. The C-terminal end of the N protein contains the major antigenic region when probed with antisera from humans and pigs infected naturally.
The contributing molecular pathways underlying the pathogenesis of breast cancer need to be better characterized. The principle of our study was to better understand the genetic mechanism of oncogenesis for human breast cancer and to discover new possible tumor markers for use in clinical practice. We used complimentary DNA (cDNA) microarrays to compare gene expression profiles of treated Michigan Cancer Foundation-7 (MCF-7) with recombinant bromelain and untreated MCF-7. SpringGene analysis was carried out of differential expression followed by Ingenuity Pathway Analysis (IPA), to understand the underlying consequence in developing disease and disorders. We identified 1,102 known genes differentially expressed to a significant degree (p<0.001) changed between the treatment. Within this gene set, 20 genes were significantly changed between treated cells and the control cells with cutoff fold change of more than 1.5. These genes are RNA-binding motif, single-stranded interacting protein 1 (RBMS1), ribosomal protein L29 (RPL29), glutathione S-transferase mu 2 (GSTM2), C15orf32, Akt3, B cell translocation gene 1 (BTG1), C6orf62, C7orf60, kinesin-associated protein 3 (KIFAP3), FBXO11, AT-rich interactive domain 4A (ARID4A), COPS2, TBPL1|SLC2A12, TMEM59, SNORD46, glioma tumor suppressor candidate region gene 2 (GLTSCR2), and LRRFIP. Our observation on gene expression indicated that recombinant bromelain produces a unique signature affecting different pathways, specific for each congener. The microarray results give a molecular mechanistic insight and functional effects, following recombinant bromelain treatment. The extent of changes in genes is related to and involved significantly in gap junction signaling, amyloid processing, cell cycle regulation by BTG family proteins, and breast cancer regulation by stathmin1 that play major roles.
This study was established to test the hypothesis of whether the codon optimization of fish growth hormone gene (FGH) based on P. pastoris preferred codon will improve the quantity of secreted rFGH in culture supernatant that can directly be used as fish feed supplements. The optimized FGH coding sequence (oFGH) and native sequence (nFGH) of giant grouper fish (Epinephelus lanceolatus) were cloned into P. pastoris expression vector (pPICZαA) downstream of alcohol oxidase gene (AOX1) for efficient induction of extracellular rFGH by adding 1% of absolute methanol. The results showed that recombinant P. pastoris was able to produce 2.80 ± 0.27 mg of oFGH compared to 1.75 ± 0.25 of nFGH in one litre of culture supernatant. The total body weight of tiger grouper fingerlings fed with oFGH increased significantly at third (P < 0.05) and fourth weeks (P < 0.01) of four-week experiment period compared to those fed with nFGH. Both oFGH and nFGH significantly enhanced the final biomass and fish survival percentage. In conclusion, codon optimization of FGH fragment was useful to increase rFGH quantity in the culture supernatant of P. pastoris that can be directly used as fish feed supplements. Further studies are still required for large scale production of rFGH and practical application in aquaculture production.
The extract from Mitragyna speciosa has been widely used as an opium substitute, mainly due to its morphine-like pharmacological effects. This study investigated the effects of M. speciosa alkaloid extract (MSE) on human recombinant cytochrome P450 (CYP) enzyme activities using a modified Crespi method. As compared with the liquid chromatography-mass spectrometry method, this method has shown to be a fast and cost-effective way to perform CYP inhibition studies. The results indicated that MSE has the most potent inhibitory effect on CYP3A4 and CYP2D6, with apparent half-maximal inhibitory concentration (IC(50)) values of 0.78 µg/mL and 0.636 µg/mL, respectively. In addition, moderate inhibition was observed for CYP1A2, with an IC(50) of 39 µg/mL, and weak inhibition was detected for CYP2C19. The IC(50) of CYP2C19 could not be determined, however, because inhibition was <50%. Competitive inhibition was found for the MSE-treated CYP2D6 inhibition assay, whereas non-competitive inhibition was shown in inhibition assays using CYP3A4, CYP1A2 and CYP2C19. Quinidine (CYP2D6), ketoconazole (CYP3A4), tranylcypromine (CYP2C19) and furafylline (CYP1A2) were ACCESSused as positive controls throughout the experiments. This study shows that MSE may contribute to an herb-drug interaction if administered concomitantly with drugs that are substrates for CYP3A4, CYP2D6 and CYP1A2.
We report a case of a patient with hypertension and ischaemic heart disease on anti-platelet treatment, who developed uniocular profound visual loss from a submacular haemorrhage secondary to valsalva retinopathy. He was treated with a combination of intravitreal recombinant tissue plasminogen activator (rtPA) and sulphur hexafluoride (SF6) gas followed by strict prone positioning. He demonstrated significant displacement of the haemorrhage and improvement of vision postoperatively.
A direct recovery of recombinant nucleocapsid protein of Nipah virus (NCp-NiV) from crude Escherichia coli (E. coli) homogenate was developed successfully using a hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC). The nucleic acids co-released with the recombinant protein have increased the viscosity of the E. coli homogenate, thus affected the axial mixing in the EBAC column. Hence, DNase was added to reduce the viscosity of feedstock prior to its loading into the EBAC column packed with the hydrophobic interaction chromatography (HIC) adsorbent. The addition of glycerol to the washing buffer has reduced the volume of washing buffer applied, and thus reduced the loss of the NCp-NiV during the washing stage. The influences of flow velocity, degree of bed expansion and viscosity of mobile phase on the adsorption efficiency of HI-EBAC were studied. The dynamic binding capacity at 10% breakthrough of 3.2mg/g adsorbent was achieved at a linear flow velocity of 178 cm/h, bed expansion of two and feedstock viscosity of 3.4 mPas. The adsorbed NCp-NiV was eluted with the buffer containing a step gradient of salt concentration. The purification of hydrophobic NCp-NiV using the HI-EBAC column has recovered 80% of NCp-NiV from unclarified E. coli homogenate with a purification factor of 12.5.
HBcAg (hepatitis B core antigen) is a nanoplex bioproduct that has a great potential in the development of therapeutic drugs and vaccines. In the present study, a continuous-flow bead milling for the disruption of Escherichia coli was optimized and a direct recovery protocol to isolate the recombinant HBcAg from the unclarified E. coli disruptate was developed. The optimal condition for continuous-flow bead milling for the release of HBcAg from E. coli was achieved at a feed flow rate of 15 litres/h, biomass concentration of 10% [ww/v (wet weight/vol.)] and impeller tip speed of 14 m/s. The sucrose-density-gradient analysis showed that the particulate form of the HBcAg released by this optimal condition is still preserved. In the direct purification of HBcAg from the unclarified disruptate, the AE-EBAC (anion-exchange expanded-bed adsorption chromatography) technique was employed. A 54% adsorption and 50.7% recovery of HBcAg were achieved in this direct recovery process. The purity of HBcAg recovered was 49.8%, which corresponds to a purification factor of 2.0. ELISA showed that the HBcAg recovered is functionally active.
We constructed recombinant phage particles displaying the Bacillus thuringiensis Cry1Ba4 active toxin using the pfUSE5 and pComb3X phagemid vectors. The recombinant phage particles were screened and evaluated for displayed biologically active Cry1Ba4 toxin against the target insect larvae. Concurrent expression of Cry1Ba4 protoxin was carried out using the pETBlue -2 plasmid expression vector in Escherichia coli Tuner (DE3)pLacI and the protoxin was successfully expressed at a size of 129 kDa. In the bioassay, 3.30 mg crude extract of Cry1Ba4 protoxin, 9.35 x 10(9) TU and 7.70 x 10(9) TU of induced recombinant phage particles carrying Cry1Ba4 active toxin displayed on pComb3X and pFUSE5, respectively, demonstrated mortality of greater than 85% against Plutella xylostella (third-instar) within 48 hours. Thus, we have successfully displayed the Cry1Ba4 activated toxin on the surface of a phage and demonstrated toxicity towards larvae.
Global resurgence of dengue virus infections in many of the tropical and subtropical countries is a major concern. Therefore, there is an urgent need for the development of successful drugs that are both economical and offer a long-lasting protection. The viral NS2B-NS3 serine protease (NS2B-NS3pro) is a promising target for the development of drug-like inhibitors, which are not available at the moment. In this study, we report retrocyclin-1 (RC-1) production in E. coli as a recombinant peptide to test against dengue NS2B-NS3pro.
Chikungunya virus (CHIKV) is a mosquito-borne arbovirus which poses a major threat to global public health. Definitive CHIKV diagnosis is crucial, especially in distinguishing the disease from dengue virus, which co-circulates in endemic areas and shares the same mosquito vectors. Laboratory diagnosis is mainly based on serological or molecular approaches. The E2 glycoprotein is a good candidate for serological diagnosis since it is the immunodominant antigen during the course of infection, and reacts with seropositive CHIKV sera. In this chapter, we describe the generation of stable clone Sf9 (Spodoptera frugiperda) cells expressing secreted, soluble, and native recombinant CHIKV E2 glycoprotein. We use direct plasmid expression in insect cells, rather than the traditional technique of generating recombinant baculovirus. This recombinant protein is useful for serological diagnosis of CHIKV infection.
The Colony Stimulating Factors (CSFs) are a family of haemopoietic hormones that likely share a common ancestral origin and stimulate white blood cell development. They display unique but overlapping biological functions and stimulate the survival, proliferation, differentiation and functional activation of granulocytes and monocytes/macrophages and their precursor cells in vitro and in vivo. Each hormone has been purified and produced in active recombinant form. Recombinant G-CSF and GM-CSF are now being used around the world in a variety of clinical situations (e.g., in conjunction with chemotherapy and bone marrow transplantation) to promote the formation and function of these leukocytes. These molecules are among the first of a new generation of biological agents that will impact enormously on clinical medicine.
Matched MeSH terms: Recombinant Proteins/therapeutic use
Hendra virus (HeV) and Nipah virus (NiV) are members of a new genus, Henipavirus, in the family paramyxoviridae. Each virus encodes a phosphoprotein (P) that is significantly larger than its counterparts in other known paramyxoviruses. The interaction of this unusually large P with its nucleocapsid protein (N) was investigated in this study by using recombinant full-length and truncated proteins expressed in bacteria and a modified protein-blotting protein-overlay assay. Results from our group demonstrated that the N and P of both viruses were able to form not only homologous, but also heterologous, N-P complexes, i.e. HeV N was able to interact with NiV P and vice versa. Deletion analysis of the N and P revealed that there were at least two independent N-binding sites on P and they resided at the N and C termini, respectively. Similarly, more than one P-binding site was present on N and one of these was mapped to a 29 amino acid (aa) C-terminal region, which on its own was sufficient to interact with the extreme C-terminal 165 aa region of P.
Conventional refolding methods are associated with low yields due to misfolding and high aggregation rates or very dilute proteins. In this study, we describe the optimization of the conventional methods of reverse dilution and affinity chromatography for obtaining high yields of a cysteine rich recombinant glycoside hydrolase family 19 chitinase from Streptomyces griseus HUT6037 (SgChiC). SgChiC is a potential biocontrol agent and a reference enzyme in the study and development of chitinases for various applications. The overexpression of SgChiC was previously achieved by periplasmic localization from where it was extracted by osmotic shock and then purified by hydroxyapatite column chromatography. In the present study, the successful refolding and recovery of recombinant SgChiC (r-SgChiC) from inclusion bodies (IB) by reverse dilution and column chromatography methods is respectively described. Approximately 8 mg of r-SgChiC was obtained from each method with specific activities of 28 and 52 U/mg respectively. These yields are comparable to that obtained from a 1 L culture volume of the same protein isolated from the periplasmic space of E. coli BL21 (DE3) as described in previous studies. The higher yields obtained are attributed to the successful suppression of aggregation by a stepwise reduction of denaturant from high, to intermediate, and finally to low concentrations. These methods are straight forward, requiring the use of fewer refolding agents compared with previously described refolding methods. They can be applied to the refolding of other cysteine rich proteins expressed as inclusion bodies to obtain high yields of actively folded proteins. This is the first report on the recovery of actively folded SgChiC from inclusion bodies.
Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM β-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of β-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. β-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant β-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular β-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly β-CGTase by the recombinant E. coli.
A stirred fluidized bed (SFB) ion exchange chromatography was successfully applied in the direct recovery of recombinant enhanced green fluorescent protein (EGFP) from the unclarified Escherichia coli homogenate. Optimal conditions for both adsorption and elution processes were determined from the packed-bed adsorption systems conducted at a small scale using the clarified cell homogenate. The maximal adsorption capacity and dissociation constant for EGFP-adsorbent complex were found to be 6.3 mg/mL and 1.3 × 10-3 mg/mL, respectively. In an optimal elution of EGFP with 0.2 M of NaCl solution (pH 9) and at 200 cm/h, the recovery percent of the EGFP was approximately 93%. The performances of SFB chromatography for direct recovery of EGFP was also evaluated under different loading volumes (50-200 mL) of crude cell homogenate. The single-step purification of EGFP by SFB recorded in a high yield (95-98%) and a satisfactory purification factor (~3 folds) of EGFP from the cell homogenate at 200 rpm of rotating speed.
Mechanical and non-mechanical breakages of bacterial cells are usually the preliminary steps in intracellular protein purification. In this study, the recombinant green fluorescent protein (GFP) was purified from intact Escherichia coli cells using preparative PAGE. In this purification process, cells disruption step is not needed. The cellular content of E. coli was drifted out electrically from cells and the negatively charged GFP was further electroeluted from polyacrylamide gel column. SEM investigation of the electrophoresed cells revealed substantial structural damage at the cellular level. This integrated purification technique has successfully recovered the intracellular GFP with a yield of 82% and purity of 95%.
Encapsidation by nucleocapsid (N) protein is crucial for viral RNA to serve as a functional template for virus replication. However, the potential region that is vital for RNA encapsidation of Nipah virus (NiV) is still unknown. Thus, this study was aimed to identify these regions using a NiV minireplicon system. A series of broad range internal deletion mutations was generated in the 5' non-translated region (NTR) of the N gene mRNA region of NiV leader promoter via site-directed overlapping PCR-mediated mutagenesis. The mutation effects on synthesis and encapsidation of antigenome RNA, transcription, and RNA binding affinity of N protein were evaluated. The deletions of nucleotides 73-108, 79-108, and 85-108 from NiV leader promoter inhibited the encapsidation of antigenome RNA, while the deletion of nucleotides 103-108 suppressed the synthesis and encapsidation of antigenome RNA, implying that these regions are required for genome replication. Surprisingly, none of the mutations had detrimental effect on viral transcription. Using isothermal titration calorimetry, the binding of NiV N protein to genome or antigenome RNA transcript lacking of nucleotides 73-108 was found to be suppressed. Additionally, in silico analysis on secondary structure of genome RNA further supported the plausible cause of inefficient encapsidation of antigenome RNA by the loss of encapsidation signal in genome template. In conclusion, this study suggests that the nucleotides 73-90 within 5' NTR of the N gene mRNA region in NiV leader promoter contain cis-acting RNA element that is important for efficient encapsidation of antigenome RNA.
Toxoplasmosis is an important cause of congenital infection. The present study was performed to evaluate the usefulness of recombinant (r) GRA-7 cloned from nucleotides (n) 39-711 in discriminating between acute and chronic toxoplasmosis. First, commercial IgM, IgG and IgG avidity ELISAs were used to determine the serological profile of the sera. Serum samples were from 20 symptomatic patients with acute infection (low IgG avidity, IgM positive), 10 with chronic infection (high IgG avidity, IgM negative) and 10 with indeterminate IgG avidity (IgM positive) which were tested for IgG avidity status with an in-house developed IgG avidity Western blot using the rGRA-7 recombinant antigen. All 20 sera from cases of probable acute infection showed bands which either faded out completely or reduced significantly in intensity after treatment with 8 M urea, whereas the band intensities of the 10 serum samples from chronic cases remained the same. Of the 10 sera with indeterminate IgG avidity status, after treatment with 8 M urea the band intensities with six sera remained the same, two sera had completely faded bands and another two sera had significantly reduced band intensities. Discrimination between acute and chronic toxoplasmosis was successfully performed by the in-house IgG avidity Western blot.