Development of Adenovirus-Based Covid-19 Vaccine Candidate in Indonesia.

Covid-19 pandemic has struck worldwide by end of 2019 and the use of various vaccine platforms was one of the main strategies to end this. To meet the needs for vaccine technology equality among many countries, we developed adenovirus-based Covid-19 vaccine candidate in Indonesia. SARS-CoV-2 Spike gene (S) was constructed into pAdEasy vector. The recombinant serotype 5 Adenovirus (AdV_S) genome was transfected into AD293 cells to produce recombinant adenovirus. Characterization using PCR confirmed the presence of spike gene. Transgene expression analysis showed the expression of S protein in AdV_S infected AD293 and A549 cells. Optimization of viral production showed the highest titer was obtained at MOI of 0.1 and 1 at 4 days. The in vivo study was performed by injecting Balb/c mice with 3.5 × 107 ifu of purified adenovirus. The result showed that S1-specific IgG was increased up to 56 days after single-dose administration of AdV_S. Interestingly, significant increase of S1 glycoprotein-specific IFN-γ ELISpot was observed in AdV_S treated Balb/c mice. In conclusion, the AdV_S vaccine candidate was successfully produced at laboratory scale, immunogenic, and did not cause severe inflammation in Balb/c mice. This study serves as initial step towards manufacturing of adenovirus-based vaccine in Indonesia.

Direct relationship between dimeric form and activity in the acidic copper-zinc superoxide dismutase from lemon.

The copper-zinc superoxide dismutase (CuZnSOD) from lemon (SOD_CL) is active in an acidic environment and resists proteolytic degradation. The enzyme occurs as a dimer, which has an indirect effect on the enzyme activity as the monomer retains only ∼35% of the activity. Here, the crystal structure of SOD_CL at 1.86 Šresolution is reported that may explain this peculiarity. The crystal belonged to space group P21, with unit-cell parameters a = 61.11, b = 74.55, c = 61.69 Å, ß = 106.86°, and contained four molecules in the asymmetric unit. The overall structure of SOD_CL resembles that of CuZnSOD from plants. The structure of SOD_CL shows a unique arrangement of surface loop IV that connects the dimer interface and the active site, which is located away from the dimer-interface region. This arrangement allows direct interaction between the residues residing in the dimer interface and those in the active site. The arrangement also includes Leu62 and Gln164, which are conserved in cytoplasmic CuZnSOD. This supports the classification of SOD_CL as a cytoplasmic CuZnSOD despite sharing the highest amino-acid sequence homology with CuZnSODs from spinach and tomato, which are chloroplastic.

Development of Chimeric Hepatitis B (HBV) - Norovirus (NoV) P particle as candidate vaccine against Hepatitis B and norovirus infection.

Introduction: Hepatitis B remains a global problem with no effective treatment. Here, a mucosal vaccine candidate was developed with HBsAg and HBcAg, to provide both prophylactic and therapeutic protection against hepatitis B. The antigens were presented using the P particle of human norovirus (HuNov). As a result, the chimeric HBV - HuNoV P particle can act as a dual vaccine for hepatitis B and HuNoV. Methods: The vaccine candidate was expressed and purified from Escherichia coli BL21 (DE3) cells. HBV-HuNoV chimeric P particles were successfully expressed and isolated, with sizes of approximately 25.64 nm. Then, the HBV-HuNoV chimeric P particles were evaluated for safety and immunogenicity in mice and gnotobiotic (Gn) pigs. After three doses (5 µg/dose in mice and 200 µg/dose in Gn pigs) of intranasal immunization, humoral and cellular immune responses, as well as toxicity, were evaluated. Results: The vaccine candidate induced strong HBV-HuNoV specific IFN-γ producing T-cell responses in the ileum, spleen, and blood of Gn pigs. Serum IgG and IgA antibodies against HBV-HuNoV chimeric P particles also increased significantly in Gn pigs. Increased HBsAg- and HuNoV-specific serum IgG responses were observed in mice and Gn pigs, although not statistically significant. The vaccine candidate did not show any toxicity in mice. Conclusions: In summary, the chimeric HBV-HuNoV P particle vaccine given intranasally was safe and induced strong cellular and humoral immune responses in Gn pig. Modifications to the vaccine structure and dosage need to be evaluated in future studies to further enhance immunogenicity and induce more balanced humoral and cellular responses.

Purification and proteomic analysis of potent fibrinolytic enzymes extracted from Lumbricus rubellus.

BACKGROUND: Lumbrokinase derived from earthworms, Lumbricus rubellus is known to have fibrinolytic enzymes that have potential as therapeutic drugs due to its ability to dissolve fibrin. The current study is aimed to purify the Lumbrokinase from L. rubellus and identify its protein component. METHODS: Water extract of local earthworm Lumbricus rubellus revealed several proteins. Therefore, to identify its protein component, purification through HiPrep DEAE fast flow and proteomic analysis were conducted prior to identifications. A combination of two-dimension gel electrophoresis (2DE) and electrospray ionization mass spectrometry analysis was used to identify the purified fractions. RESULTS: The purified fractions contain five protein bands, namely F25-1, F25-2, F85-1, F85-2, and F85-3, which displayed strong fibrinogenolytic activity. F25 fractions showed fibrinogenolytic activity of 974.85 U/mg, while F85 fractions showed higher activity of 1,484.11 U/mg. Fractions F85-1, F85-2, and F85-3 showed molecular weights of 42.6 kDa, 27.03 kDa, and 14 kDa, respectively and were identified as Lumbrokinase iso-enzymes. CONCLUSION: This preliminary study indicates that the F25 and F85 fractions are similar to published fibrinolytic protease-1 and lumbrokinase, respectively, in terms of their amino acid sequence.

Introducing Intermolecular Interaction to Strengthen the Stability of MnSOD Dimer.

Manganese superoxide dismutase from Staphylococcus equorum (MnSODSeq) maintains its activity upon treatments like a wide range of pH, addition of detergent and denaturing agent, exposure to ultraviolet light, and heating up to 50 °C. The enzyme dimer dissociates at 52-55 °C, while its monomer unfolds at 63-67 °C. MnSOD dimeric form is indispensable for the enzyme activity; therefore, strengthening the interactions between the monomers is the most preferred strategy to improve the enzyme stability. However, to date, modification of MnSODSeq at the dimer interface has been unfruitful despite excluding the inner and outer sphere regions that are important to the enzyme activity. Here, a new strategy was developed and K38R-A121E/Y double substitutions were proposed. These mutants displayed similar enzyme activity to the wild type. K38R-A121E dimer was thermally more stable and its monomer stability was similar to the wild type. The thermal stability of K38R-A121Y dimer was similar to the wild type but its monomer was thermally less stable. In addition, the structure of the previously reported L169W mutant was also elucidated. The L169W mutant structure showed that intramolecular modification can decrease flexibility of the MnSODSeq monomer and leads to a less stable enzyme with similar activity to the wild type. Thus, while the enzyme activity depends on arrangement of residues in the dimer interface, the stability appears to depend more on its monomeric architecture. Furthermore, in the L169W structure in complex with azide, which is a specific inhibitor for MnSOD, one of the azide molecules was present in the dimer interface region that previously has been identified to involve in the enzymatic reaction. Nevertheless, the present results show that an MnSODSeq mutant with better thermal stability has been obtained.

The role of S126 in the Staphylococcus equorum MnSOD activity and stability.

The dimeric form of manganese superoxide dismutase is instrumental for activity because each of the monomers provides amino acid residues participating in the enzymatic reaction. Hence, preventing dissociation of the dimer would maintain the enzymatic activity in detrimental conditions e.g. high temperature. To prevent dissociation of the dimer, a disulphide (S-S) bond was introduced at the dimer interface. In the wild type structure, S126 interacts with S126 of the other monomer. In the presented work, a mutant was designed with an S126C substitution. The crystal structure of the S126C mutant showed that only 50-70% of monomers formed the S-S bond. This observed imperfect S-S bonding was likely caused by photolytic S-S bond breakage mediated by the neighbouring tryptophan residue. In the wild type, S126 is located facing W163 and forms a water-mediated hydrogen bond with E164; W163 and E164 are crucial in the enzyme's activity. The replacement of S126 by a cysteine residue lowered the activity of the enzyme by ~70%. S126 has never been considered to play a role in the enzyme's activity or stability, thus the finding showed the importance of this residue.

Molecular Characterization of Bacterial Fibrinolytic Proteins from Indonesian Traditional Fermented Foods.

Previously, the crude extracts of recombinant Nattokinase (NK) variants i.e. NatTK and NatOC and one wild type Douchi Fibrinolytic Enzyme (DFE) from Indonesian traditional fermented foods has been shown to demonstrate fibrinolytic activity. Both NKs contain substitutions of D41N, V192A and 252-RLQHTLEALSTM-263 but NatOC has additional V4F. In the present study, the effects of amino acid substitutions in NK variants and G169A in DFE on their enzyme characteristics were evaluated. Pure proteins were obtained using two sequential steps chromatography using ion exchange and a gel filtration columns. Their activities were determined with fibrin plate, fibrin zymography, fibrinogen hydrolysis, and chromogenic assays. The fibrinogen degradation profile of the wild type NK (NatWT) was different to the NK variants but similar to DFEs. Optimum activity of all the NKs and DFEs was achieved at 50 °C while the optimum pH for NatWT/DFEs and NK variants were 8 and 7, respectively. DFEG169A exhibited higher fibrinogen degradation rate and fibrin specific activity than DFE. PMSF inhibited all the NKs and DFEs while SDS and EDTA caused lower activity. The NK variants were more resistant towards Na+ and Ca2+ but more sensitive to K+. The amino acid substitutions in NK variants alter their fibrinogen degradation profile, optimum working pH, working pH range, and resistance to some ions. Substitutions in NK variants likely promote structural changes, particularly with the binding mode of the calcium ion cofactor. The results provide a beneficial basis for future development of fibrino(gen)olytic proteins with improved properties for cardiovascular diseases therapy.

Construction and Characterization of a Medium Copy Number Expression Vector Carrying Auto-Inducible dps Promoter to Overproduce a Bacterial Superoxide Dismutase in Escherichia coli.

Medium copy number expression vector and auto-inducible promoter could be a solution for producing recombinant therapeutic proteins in industrial scale regarding plasmid stability, cost, and product quality. This work aimed to construct a medium copy number pBR322-based expression vector carrying auto-inducible promoter, determine its ability to express heterologous gene, and study its segregational stability. Three stationary-phase promoters of Escherichia coli genes (gadA, dps and sbmC) were used to produce a superoxide dismutase from Staphylococcus equorum (rMnSODSeq) coding region from pBR322Δtet (pBR322-mini). Four plasmids were constructed with different promoters, i.e., T7 (pBMsod), gadA (pMCDsod), dps (pCADsod), and sbmC (pCDSsod) using pBR322-mini as backbone. Results showed that rMnSODSeq expression from pBMsod was significantly higher than that from pJExpress414sod (high copy number plasmid). Meanwhile, rMnSODSeq from pCADsod (auto-inducible promoter) was as high as from pBMsod (IPTG-inducible T7 promoter). rMnSODSeq expressed from pCADsod when bacterial cells entered stationary phase appeared as an active protein band of 23.5 kDa when analyzed by zymography and SDS-PAGE. pCADsod displayed the highest stability compared with pBMsod and pJEXpress414sod by plasmid retention assay. We demonstrate the use of an auto-inducible dps promoter to express high level of heterologous protein, an SOD of S. equorum, from a stable expression vector with medium copy number.

Leu169Trp substitution in MnSOD from Staphylococcus equorum created an active new form of similar resistance to UVC irradiation.

Manganese superoxide dismutase from Staphylococcus equorum (MnSODSeq) maintains its activity after up to 45 minutes of UVC radiation. The enzyme occurs in a dimeric form that likely contributes to its activity and stability. Therefore, maintaining the dimeric form could be a way to improve the enzyme's stability. One of the main interactions for dimer formation occurs between Tyr168 and His31, of which the latter is also involved in the enzymatic reaction. UVC radiation may cause alterations in the electronic structure of the phenolic ring in the Tyr168 side chain: this may disrupt the Tyr168-His31 pairing and lead to enzyme instability and/or activity loss. In this report, a Leu169Trp substitution was carried out to protect the Tyr168 residue by introducing an amino acid with an aromatic side chain for better photon absorption of the UV light. Interestingly, although the substitution appeared to have a minor effect on enzyme stability and activity upon UVC irradiation, the melting temperature (TM) of the Leu169Trp mutant was different. Unlike the native protein, the TM of the mutant had not changed after UV irradiation. Thus, our effort to extend the resistance to UVC radiation was not successful, but we have discovered a biologically active new form. The present finding provides evidence that MnSODSeq maintains most of its activity and resistance to UVC irradiation as long as the dimer and its glutamate-bridge are intact, despite an alteration that destabilizes its monomeric structure. The present finding further unravels the relationship between the structure of the enzyme and its activity. Furthermore, the results may provide further insight in how to modify the enzyme to improve its characteristics for application in medicine or cosmetics.

Gliadin Peptide Facilitates FITC Dextran Transport across the Non Everted Gut Sac of Rat Small Intestine.

Superoxide dismutase (SOD) is an antioxidant protein. When administered orally, it has low bioavailability due to its low permeation. In a previous study we fused gliadin peptide P51 (LGQQQPFPPQQPYPQPQPF) and gliadin peptide P61 (QQPYPQPQPF) with SOD Citrus limon (SOD_Cl), namely GliSOD_P51 and GliSOD_P61 to increase permeation of SOD_Cl through intestine. In this work, the permeation of fluorescein isothiocyanate (FITC)-Dextran 10 kDa, FD10 and 40 kDa, FD40 as paracellular transport markers across excised rat intestinal wall was investigated with the presence of GliSOD_P51 and GliSOD_P61. A permeability study was performed using non-everted rat intestine by incubating FD10 or FD40 with SOD_Cl, and GliSOD_P61. The presence of SOD_Cl, GliSOD_P51 or GliSOD_P61 inside intestine (apical) and outside intestine (basolateral) was analyzed by protein electrophoresis. The concentration of FD that penetrated to the basolateral solution was analyzed by spectrofluorometry. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed the presence of GliSOD_P51 and GliSOD_P61 but not SOD_Cl in basolateral compartment. The percentage of FD10 but not FD40 and SOD_Cl that penetrated to the basolateral solution significantly increased with the presence of gliadin in GliSOD_P51 and GliSOD_P61. GliSOD_P51 and GliSOD_P61 are able to penetrate the rat intestinal epithelial membrane and the gliadin peptides facilitate FD10 to penetrate the epithelial.

The first crystal structure of manganese superoxide dismutase from the genus Staphylococcus.

A recombinant Staphylococcus equorum manganese superoxide dismutase (MnSOD) with an Asp13Arg substitution displays activity over a wide range of pH, at high temperature and in the presence of chaotropic agents, and retains 50% of its activity after irradiation with UVC for up to 45 min. Interestingly, Bacillus subtilis MnSOD does not have the same stability, despite having a closely similar primary structure and thus presumably also tertiary structure. Here, the crystal structure of S. equorum MnSOD at 1.4 Šresolution is reported that may explain these differences. The crystal belonged to space group P3221, with unit-cell parameters a = 57.36, b = 57.36, c = 105.76 Å, and contained one molecule in the asymmetric unit. The symmetry operation indicates that the enzyme has a dimeric structure, as found in nature and in B. subtilis MnSOD. As expected, their overall structures are nearly identical. However, the loop connecting the helical and α/ß domains of S. equorum MnSOD is shorter than that in B. subtilis MnSOD, and adopts a conformation that allows more direct water-mediated hydrogen-bond interactions between the amino-acid side chains of the first and last α-helices in the latter domain. Furthermore, S. equorum MnSOD has a slightly larger buried area compared with the dimer surface area than that in B. subtilis MnSOD, while the residues that form the interaction in the dimer-interface region are highly conserved. Thus, the stability of S. equorum MnSOD may not originate from the dimeric form alone. Furthermore, an additional water molecule was found in the active site. This allows an alternative geometry for the coordination of the Mn atom in the active site of the apo form. This is the first structure of MnSOD from the genus Staphylococcus and may provide a template for the structural study of other MnSODs from this genus.

Evaluation of alum-based adjuvant on the immunogenicity of salmonella enterica serovar typhi conjugates vaccines.

The function of adjuvant in maintaining the long-term immune response to Typhoid conjugate vaccine (TCV) was evaluated in. Two TCV products, Vi-DT and Vi-TT, were formulated in either aluminum phosphate (AlPO4) or aluminum hydroxide (AlOH) as adjuvants and TCV formulated in phosphate buffer saline were used as controls. In each case, a group of Balb/c mice was injected intramuscularly with two doses of the formulated vaccine at two-week intervals. The anti-Vi IgG responses were monitored by Enzyme-Linked Immunosorbent Assay and the levels of CD4+ T-cells expressing cytokine were characterized using intracellular cytokine staining. All mice immunized by TCV formulated in adjuvant elicited anti-Vi response to a higher level than the group receiving TCV formulated in PBS. The extent of adsorption of TCV in AlOH was greater than that in AlPO4, and this finding correlated well with the observation that the mice immunized with two doses of Vi-DT(AlOH) elicited anti-Vi IgG to a level higher than that seen with Vi-DT(AlPO4). The mice primed with Vi-TT(AlOH) produced lower anti-Vi IgG (25.901 GM) compared to those receiving Vi-TT(AlPO4) (49.219 GM). However, after the second injection, the former raised the antibody level significantly to 137.008 GM while the latter provided a value of only 104.966 GM. The groups of mice vaccinated by TCV formulated in AlOH expressed IL4 at higher levels than the other groups, which correlated positively with the high Anti-Vi IgG in these animals. In conclusion, AlOH could be recommended as an effective adjuvant for TCV to provide a long-term immune response.

The light subunit of mushroom Agaricus bisporus tyrosinase: Its biological characteristics and implications.

The light subunit of mushroom Agaricus bisporus tyrosinase (LSMT) is a protein of unknown function that was discovered serendipitously during the elucidation of the crystal structure of the enzyme. The protein is non-immunogenic and can penetrate the intestinal epithelial cell barrier, and thus, similar to its structural homologue HA-33 from Clostridium botulinum, may be potentially absorbable by the intestine. LSMT also shares high structural homology with the ricin-B-like lectin from the mushroom Clitocybe nebularis (CNL), which has been shown to display biological activity against leukemic cancer cells and dendritic cells. Therefore, we evaluated the biological activity of LSMT. An in vitro assay suggested that LSMT presentation to most of the cancer cell lines studied has a negligible effect on their proliferation. However, inhibition of cell growth and a slight stimulation of cell proliferation were observed with breast cancer and macrophage cells, respectively. LSMT appeared to be relatively resistant against proteolysis by trypsin and papain, but not bromelain. Challenges with gastric and intestinal juice suggested that the protein is resistant to gastrointestinal tract conditions. This is the first report on the biological characteristics and implication of LSMT.

Structure-activity relationship of a recombinant hybrid Manganese superoxide dismutase of Staphylococcus saprophyticus/S. equorum.

Recombinant hybrid Manganese superoxide dismutase from Staphyloccus saphropyticus/S. equorum (rMnSODSeq) exhibits stability at high temperatures. The enzyme occurs as a dimer that dissociates around 52°C prior to unfolding of the monomer around 64°C, demonstrating contribution of the dimeric form to stability. Here, structure - activity relationship of rMnSODSeq was evaluated on the basis of its activity and stability in the presence of inhibitors, NaCl, denaturants, detergents, reducing agents, and at different pH values. The activity was evaluated at both 37°C and 52°C, which the latter is the temperature for dissociation of the dimer. Dimer to monomer transition coincided with significant decrease in residual activity at 52°C. However, the activity assay results at 52°C and 37°C suggest spontaneous re-association of the monomer into dimer. Intriguingly, various new species with melting temperature (TM) values other than those of the dimer or monomer were observed. These species displayed medium to comparable level of residual activities to the native at 37°C. This report suggests that dimer to monomer transition may be not the only explanation for activity loss or decrease.

Separate Toxin-Antidote Stabilization System in Two Plasmids for Recombinant Protein Production.

BACKGROUND: Expression vector is an important component in the production of therapeutic recombinant proteins. Most of the commercialized expression vectors apply antibiotic-based selection system. Meanwhile, World Health Organization highly recommends for the alternative system due to its potentials to cause spreading of resistance gene and hypersensitivity to some people. METHODS: In current work, we developed an expression system for Escherichia coli using the toxinantidote system in two separated plasmids. An antidote gene (ccdA) with its natural promoter and terminator was constructed in a plasmid (pDCSAsod) containing a DNA fragment encoding recombinant superoxide dismutase from Staphylococcus equorum (rMnSODSeq) as a model. The gene expression was directed under T7 promoter and regulated by lac operator. The toxin gene (ccdB) was located in a separate plasmid (pDCSB) under PBAD promoter. This study aimed to study the growth profile of the host in the presence of both plasmids, to determine plasmids stability, and the effect of the toxinantidote system on rMnSODSeq production and activity. RESULTS: The presence of both plasmids did not affect the growth profile of E. coli BL21(DE3), while the plasmid stability was 94% for pDCSAsod and 68% for pDCSB at the end of protein production time. The yield of purified rMnSODSeq was 3.2 mg/ml and the enzyme was shown to be active by a zymography assay. CONCLUSION: In conclusion, for the first time, we show that toxin-antidote system in two separated plasmids has the potential for the production of recombinant therapeutic proteins and is more flexible in choosing the E. coli strain compared to established chromosomally integrated toxin-antidote selection system.

Study of HMG-CoA Reductase Inhibition Activity of the Hydrolyzed Product of Snakehead Fish (Channa striata) Skin Collagen with 50 kDa Collagenase from Bacillus licheniformis F11.4.

Bioactive peptides produced from enzymatic hydrolysis fibrous protein have been proven to have several biological activities. Previous study showed that the hydrolysis product of snakehead fish skin collagen with 26 kDa collagenase from Bacillus licheniformis F11.4 showed HMG-CoA (HMGR) inhibition activity. The aim of this research was to determine the ability of the hydrolysis product produced from snakehead fish skin collagen hydrolysed by 50 kDa collagenase from B. licheniformis F11.4 in inhibiting HMGR activity. Snakehead fish skin collagen was extracted using an acid method and collagenase was produced from B. licheniformis F11.4 using half-strength Luria Bertani (LB) medium containing 5% collagen. Crude collagenase was concentrated and fractionated using the DEAE Sephadex A-25 column eluted with increasing gradient concentrations of NaCl. Collagen, collagenase, and fractions were analyzed using SDS-PAGE and collagenolytic activity was analyzed by the zymography method. Collagenase with 50 kDa molecular weight presented in fraction one was used to hydrolyze the collagen. The reaction was done in 18 hours at 50°C. The hydrolysis product using 3.51 µg collagen and 9 ng collagenase showed 25.8% inhibition activity against pravastatin. This work shows for the first time that the hydrolysis product of snakehead fish skin collagen and 50 kDa collagenase from B. licheniformis F11.4 has potential as an anticholesterol agent.

Plasmid Copy Number Determination by Quantitative Polymerase Chain Reaction.

Recombinant therapeutic proteins are biopharmaceutical products that develop rapidly for years. Recombinant protein production in certain hosts requires vector expression harboring the gene encoding the corresponding protein. Escherichia coli is the prokaryote organism mostly used in recombinant protein production, commonly using a plasmid as the expression vector. Recombinant protein production is affected by plasmid copy number harboring the encoded gene, hence the determination of plasmid copy number also plays an important role in establishing a recombinant protein production system. On the industrial scale, a low copy number of plasmids are more suitable due to their better stability. In the previous study we constructed pCAD, a plasmid derived from the low copy number pBR322 plasmid. This study was aimed to confirm pCAD's copy number by quantitative polymerase chain reaction (qPCR). Plasmid copy number was determined by comparing the quantification signal from the plasmid to those from the chromosome. Copy number was then calculated by using a known copy number plasmid as a standard. Two pairs of primers, called tdk and ori, were designed for targeting a single gene tdk in the chromosome and a conserved domain in the plasmid's ori, respectively. Primer quality was analyzed in silico using PrimerSelect DNASTAR and PraTo software prior to in vitro evaluation on primer specificity and efficiency as well as optimization of qPCR conditions. Plasmid copy number determination was conducted on E. coli lysates harboring each plasmid, with the number of cells ranging from 10(2)-10(5) cells/µL. Cells were lysed by incubation at 95ºC for 10 minutes, followed by immediate freezing at -4°C. pBR322 plasmid with the copy number of ~19 copies/cell was used as the standard, while pJExpress414-sod plasmid possessing the high copy number pUC ori was also determined to test the method being used. In silico analysis based on primer-primer and primer-template interactions showed that both primer pairs were acceptable and were predicted to have good performance. Those predictions were in agreement with the in vitro test that gave a single band in the PCR product's electropherogram and a single peak in DNA amplicon's melting curve with a Tm value of 79.01 ± 0.11°C for the tdk primer and 81.53 ± 0.29°C for the ori primer. The efficiency of each primer was 1.95 and 1.97, respectively. The calculation result of pCAD's copy number was 13.1 ± 0.3 copies/cell, showing that pCAD's low copy number has been determined and confirmed. Meanwhile, it was 576.3 ± 91.9 copies/cell for pJExpress414-sod, in accordance with the hypothesis that pUC ori regulates the high copy number plasmid. In conclusion, the designed primers and qPCR conditions used in this study can be used to determine plasmid copy number for plasmids with pBR322 and pUC ori. The method should be tested further on plasmids harboring other type of ori.

Removing a Cystein Group On Interferon Alpha 2b at Position 2 and 99 does Not Diminish Antitumor Activity of the Protein, Even Better.

Interferon alpha 2b is the only standard therapeutic protein for hepatitis virus infections. Further study demonstrated that this protein also posseses antitumor activity in several cancerous organs. One main pathway of this antitumor activity is mediated through antiproliferation as well as proapoptotic effects. Previously, we have successfully developed recombinant human interferon alpha 2b (rhIFNα2b) by using a synthetic gene. In addition, two mutein forms of rhIFNα2b were generated to improve the characteristics of this protein. Two point mutations showed better pharmacokinetic profiles than one point mutation as well as the native form. In the present study, this mutein form was studied for ist antitumor effect in vitro using HepG2 cells. As a comparison, the native form as well as a commercial rIFNα2b were used. Several parameters were investigated including the MTT assay, cell viability test, cell cycle using flow cytometric analysis, and the genes and protein expressions involved in cell growth. The latest was observed to study the mechanism of rhIFNα2b. There was no significant difference in the MTT assay and cell viability after cells were treated with both forms of rhIFNα2b. However, the mutein rhIFNα2b tended to show better proapoptotic activity reflected by flow cytometric data, protein expression of pSTAT1, and DNA expression of caspase 3.

Construction and Periplasmic Expression of the Anti-EGFRvIII ScFv Antibody Gene in Escherichia coli.

In the previous study, we constructed an expression vector carrying the anti-EGFRvIII scFv antibody gene with VH-linker-VL orientation. The proteins were successfully produced in the periplasmic space of Escherichia coli. In this study, we substituted the inserted DNA with VL-linker-VH orientation of the anti-EGFRvIII scFv gene and analyzed its expression in E. coli. The DNA fragment was amplified from its cloning vector (pTz-rscFv), subsequently cloned into a previous expression vector containing the pelB signal sequence and his-tag, and then transformed into E. coli TOP10. The recombinant plasmids were characterized by restriction, PCR, and DNA sequencing analyses. The new anti-EGFRvIII scFv antibody proteins have been successfully expressed in the periplasmic compartment of E. coli Nico21(DE3) using 0.1 mM final concentration of IPTG induction. Total proteins, soluble periplasmic and cytoplasmic proteins, solubilized inclusion bodies, and extracellular proteins were analyzed by SDS-PAGE and Western Blot analyses. The results showed that soluble scFv proteins were found in all fractions except from the cytoplasmic space.

In Silico Study to Develop a Lectin-Like Protein from Mushroom Agaricus bisporus for Pharmaceutical Application.

A lectin-like protein of unknown function designated as LSMT was recently discovered in the edible mushroom Agaricus bisporus. The protein shares high structural similarity to HA-33 from Clostridium botulinum (HA33) and Ricin-B-like lectin from the mushroom Clitocybe nebularis (CNL), which have been developed as drug carrier and anti-cancer, respectively. These homologous proteins display the ability to penetrate the intestinal epithelial cell monolayer, and are beneficial for oral administration. As the characteristics of LSMT are unknown, a structural study in silico was performed to assess its potential pharmaceutical application. The study suggested potential binding to target ligands such as HA-33 and CNL although the nature, specificity, capacity, mode, and strength may differ. Further molecular docking experiments suggest that interactions between the LSMT and tested ligands may take place. This finding indicates the possible use of the LSMT protein, initiating new research on its use for pharmaceutical purposes.