فهرست مطالب

Biomacromolecular Journal - Volume:7 Issue: 2, Autumn 2021
  • Volume:7 Issue: 2, Autumn 2021
  • تاریخ انتشار: 1402/02/04
  • تعداد عناوین: 12
  • Mohamad Ehsan Madadi, Zahra Amini-Bayat *, Bahareh Bahmani, Narges Tafreshi, Mehrdad Hashemi, Neda Mousavi Niri Pages 70-76
    The method of choice for prevention and treatment of infection with Human Papillomavirus (HPV) and consequently cervical cancer is the application of prophylactic and therapeutic HPV-vaccine. The present study aimed to clone the most antigenic epitopes of the E6 antigen and to express it in E. coli at the lab-scale.
    The sequence of immune-bioinformatically determined epitopes of E6 was synthesized in the pGH vector. The new E6 gene was cloned into the pET28 vector by double-digestion of vector and target with NcoI and XhoI restriction enzymes. The recombinant vector was transformed into DH5α and cloned E6 gene was confirmed by colony PCR and DNA sequencing. pET28 was then extracted from DH5α and transferred into the BL21(DE3) expression host. Expression optimization was performed using various parameters.
    Cloning was confirmed by colony PCR and sequencing and optimized expression was performed at 25°c, IPTG=0.1 mM, OD600=1. Due to the protein production in the form of inclusion bodies and unavailability of His-Tags, the recovered protein didn’t confirm by western blot and didn’t purify by Ni-NAT affinity chromatography.
    This study aimed to express multi-epitope recombinant protein composed of selective E6 protein epitopes in the E. coli prokaryotic expression system to achieve an effective vaccine against HPV. The produced protein might be used as a therapeutic vaccine or as a platform for HPV detection.
    Keywords: Human papilloma virus, Recombinant protein, Vaccine, E6, Antigen
  • Robabeh Jafari, Ahmad Homaei *, AliReza Ahmadi, Ehsan Kamrani Pages 77-85

    The marine environment has been recognized as a source of diverse natural compounds. Astaxanthin is the main carotenoid in crustaceans, including shrimp. Shrimp waste is considered a cheap source of natural astaxanthin. A study was performed to compare the extraction yield of astaxanthin in Penaeus semisulcatus and Penaeus merguiensis waste using sunflower and sesame oils. Furthermore, the Box-Behnken design optimized the astaxanthin extraction conditions of sunflower oil from Penaeus semisulcatus waste. The results showed that the extraction yield of astaxanthin with sunflower oil from Penaeus semisulcatus wastes (13.43 ± 0.17 μg/g) was significantly different (p <0.001) compared to sesame oil and Penaeus merguiensis. Moreover, it was shown that the maximum yield of astaxanthin extracted (14.74 ± 1.6 μg/g of waste) with sunflower oil from Penaeus semisulcatus waste was produced under ideal circumstances comprising heating duration of 120 minutes, oil: waste ratio (v/w) of 2, the heating temperature of 70 °C, and waste size 80 mesh. A regression equation for astaxanthin yield was obtained as a function of heating temperature, time of heating, oil-to-waste ratio, and size of shrimp waste.

    Keywords: Penaeus semisulcatus, Penaeus merguiensis, Astaxanthin, Box-Behnken Design
  • Mehdi Mohammadpour-Aghdam, Ahmad Molaeirad *, Azadeh Azizi Pages 86-92
    hormone as a drug product is not limited to its application to the topic of short-term treatment of pediatric patients is made recombinant. Since the low expression of this heavy protein in the recombinant state influences its effective extraction, various changes have been made on this protein in the culture and expression stage. In this study, optimization based on the concentration of DMSO additive in a culture medium, incubation temperature, and induction concentration have been investigated. The results of PAGE-SDS showed a good percentage for 1% dimethyl sulfoxide and suitable temperature for incubation of 16 ° C and an appropriate concentration for Isopropylthiogalactoside inducer 0.1 mM. In the next step, the culture specimen was optimized with 10 recombinant proteins. To ensure the correct cultivation and optimization of recombinant growth hormone, the secondary protein structure of the protein has been verified and verified using Circular Dichroism Spectroscopy (CD) and subsequently verified using the intrinsic fluorescence technique of the third protein structure.
    Keywords: Growth hormone, Additive, Dimethyl sulfoxide, Isopropyl thiogalactoside, Rotational duplex
  • Fatemeh Karami *, Saman Hosseinkhani Pages 93-102
    Higher rates of protein expression lead to the accumulation of proteins as inclusion bodies. In contrast, the expression of soluble proteins needs to be optimized to reduce the amount of accumulated proteins. Therefore, obtaining a high amount of the desired his- tag recombinant proteins always requires a balance between expression rate and accumulation. Inducer concentration, time, and temperature of induction are the main variable in the level of soluble protein or inclusion bodies expression. In summary, if the higher concentrations of the protein are toxic or tend to be aggregated inside cells, higher ODs, lower IPTG concentrations, and shorter expression times can be effective in formation of inclusion bodies. The standard purification process can also be optimized based on the Physicochemical properties, such that, in addition to increasing yield in the expression step, the activity and concentration of the desired protein is maintained in the purification step. In this study, we obtained the results of extraction of three different peptides MiRGD, GNH and HNH, and two proteins firefly mutant red-emitter luciferase, and human DT-diaphorase proteins by altering these variables on expression as well as purification conditions. Optimization of the protocols in Ni-NTA affinity chromatography of recombinant proteins with denaturants for MiRGD, GNH and HNH brought about with increase of purified protein concentrations from 0.21, 0.4, and 0.16 mg/ml in the initial attempt to 2.9, 3.4 and 2.6 mg/ml in the optimized experiment respectively. Non-denaturing optimization of purification for mutant luciferase and human diaphorase produced 4.1 and 2.6 mg/ml, respectively.
    Keywords: Inclusion body, Expression, Recombinant Proteins, Ni-NTA
  • Mahtab Jahanshah Talab, MohammadReza Housaindokht, Zeinab Tavassoli, Bijan Ranjbar * Pages 103-113

    A layer of proteins covers the nanoparticles by exposure to the nanoparticles in biological fluids. The phenomenon known as biocorona can alter the nanoparticle's specific purpose. Since the usage of nanoparticles in biological issues such as drug delivery, biosensor, and photothermal has increased, evaluating nanoparticles' interactions with biomolecules is necessary. Hydrophilic polymers form a layer of water molecules that reduces the interaction potential between nanoparticles and biomolecules. In this study, the effects of gold nanoparticle morphology (spheres and rods) and surface functionalization (PEG and GSH) on glycosylated serum albumin, a model protein in diabetic patients, its secondary structure and chemical stability using circular dimorphism technique were investigated.PEG-AuNPs exhibit a larger hydrodynamic radius than GSH-AuNPs due to their large hydrophilic tails. After interaction with the protein, the nanoparticles showed less interaction with the protein due to the formation of a layer of water around them that reduces the probability of interaction with proteins. The circular dichroism also exhibits that different modified nanoparticles do not significantly influence secondary structure. The presence of nanoparticles with hydrophiliccoatings, mainly polyethylene glycol with high water retention, reduce protein availability from urea in the long run and as a result will increase the 〖∆G〗_H2O value.

    Keywords: Biocorona, Circular dichroism, nanomaterial
  • Mojtaba Mortezavi, Mehdi Forouzesh, Abdorrasoul Malekpour *, Abdolkhalegh Keshavarzi, Reza Mohammadi, Farzaneh Kargar, Reza Deghani Pages 114-125
    The 2019 novel coronavirus (2019-nCoV/SARS-CoV-2) initially appeared as part of an important prevalence of respiratory disease centered in Hubei province, China. Now, it is a pandemic globally and is a significant public health concern. Taxonomically, SARS-CoV-2 was revealed to be a Beta coronavirus (lineage B) related to SARS-CoV and SARS-related bat coronaviruses closely, and it has been stated to have a similar receptor with SARS-CoV (ACE-2). Here, we carried out the codon usage bias (CUB) by analyzing the codon bias index (CBI), codon adaptation index (CAI), and an effective number of codons (ENC) besides phylogenetic analysis of Coronaviridae and also structural modeling of the SARS-CoV-2 spike glycoprotein. We observed that 2019-nCoV has a rich composition of AT nucleotides that strongly affects its codon usage, which seems to be not optimized for the human hosts. Moreover, a close evolutionary phylogenetic relationship was detected between SARS-CoV-2/human/IRIN/ and SARS-CoV-2/human/CHN/WH-09/2020. By in silico modeling of spike glycoprotein, an I-TASSER server, the 3Dstructure of it was also evaluated. This type of analysis would be beneficial for exploring a virus adaptation to host, and evolution and is therefore of value to developing vaccine design and pharmaceutical agents.
    Keywords: Computational Biology, SARS-CoV-2, Beta coronavirus, Codon usage, Modeling
  • Mojtaba Mortezavi *, Masood Torkzadeh-Mahani, Ali Riahi-Madvar, Mehdi Rahimi, Mahmood Maleki, Safa Lotfi Pages 126-137
    Codon usage and rare codons have mixed results on the protein structure and function. An increasing amount of data is shown that replacing the rare codons with frequently synonymous ones has diverse results as a decrease in a protein’s specific activity, changing the folding pathway, and reducing protein solubility. In this study, we investigated the situation of codon usage of the Lampyridae family luciferases using computational databases. For this, the codon feature of these luciferases was studied, bioinformatically. Also, in silico analyses of this enzyme were conducted by structural modeling on the I-TASSER web server. The status of these rare codons in these structural models was studied using SPDBV and PyMOL software. Finally, the binding site properties were studied using the AutoDock Vina. Using molecular modeling, two rare codons (Arg533 and Arg536) were analyzed that may have a critical role in the structure and function of these luciferases. AutoDock Vina was used in molecular docking that recognizes some residues that yield closely related to luciferyl-adenylate binding sites. These analyses created a new understanding of the sequence and structure of these luciferases, and our findings can be used in some fields of clinical and industrial biotechnology. This bioinformatics analysis plays an essential role in the design of new drugs.
    Keywords: luciferase, Codon bias, Rare codon, Lampyridae, Molecular Docking
  • Foruzan Moradi, Pouya Sadeghizadeh, Farhood Najafi, Majid Sadeghizadeh * Pages 138-148
    Tamoxifen (TAM) is routinely used for the treatment of estrogen-positive breast carcinoma. Approximately 40% of patients with metastatic breast cancer will develop resistance to TAM. TAM therapeutic failure has been a major challenge in the treatment of TAM-resistant breast cancer cells. Therefore, finding a way to eliminate TAM resistance is very valuable. Curcumin is a polyphenol extracted from the rhizomes of Curcuma longa and has extensive biological and pharmacological effects on many cancers. The purpose of this study was to look into the effects of dendrosomal nano-curcumin (DNC) on cell growth and apoptosis, as well as the effects of DNC on the expression levels of long non-coding RNA CCAT2 in TAM-resistant MCF-7 cells (TAM-R).
    TAM-R cells were created, and CCAT2 expression was evaluated in TAM-R compared to TAM-sensitive MCF-7 cells (TAM-S). Forty eighth hours after TAM-R treatment with 20 μM of DNC, Q-RT-PCR, and flow cytometry cell cycle and Annexin V-PI assays were performed. P-value < 0.05 was defined as statistical significance.
    CCAT2 was significantly upregulated in TAM-R compared to TAM-S. DNC administration downregulated CCAT2 expression, and markedly suppressed cell cycle and induced apoptosis in TAM-R. Furthermore, DNC decreased the anti-apoptosis gene (BCL-2) and increased the apoptotic gene (BAX) expression levels respectively in TAM-R.
    DNC promoted cell cycle arrest and apoptosis, eventually by CCAT2 downregulation in TAM-R. However, the probable mechanisms of how DNC affects CCAT2 expression levels are unknown and need future studies.
    Keywords: Dendrosomal nano-curcumin, CCAT2, Tamoxifen-resistant MCF-7, Cell cycle, Apoptosis
  • Akram Najaran, Adeleh Divsalar *, AliAkbar Saboury, Nasim Hayati-Roodbary Pages 149-158

    In pharmaceutical field, the effect of drugs on biological substances is of particular importance. Therefore, in this research, the interaction between human plasma carrier protein of Albumin (HSA) and a newly synthesized complex of 2,2′-Bipyridine-3,3′-dicarboxylic acid Oxalato Palladium(II) was tested by using site marker competitive tests and various spectroscopic methods such as ultraviolet, fluorescence, and circular dichroism (CD) at different temperatures of 25 and 37 °C. The study of fluorescence spectroscopy showed that this complex reduced the inherent fluorescence emission of HSA, which was eventually quenched by the dynamic mechanism. As well, binding constants were calculated on the palladium compound. According to the obtained thermodynamic parameters of ΔHo and ΔSo values, it was shown that the interaction of palladium complex to the protein occurs via hydrophobic force. The analysis of the circular dichroism (CD) spectrum also showed that the palladium complex caused no significant changes in the secondary structure of the protein. The results of thermal stability of the protein in the presence of palladium complex showed a decreased in thermal stability of HSA.Additionally, by the use of spectroscopic methods in the competitive experiments to identify the complex binding site on HSA using site marker competitive of warfarin for Sudlow’s site I, ibuprofen for Sudlow’s site II, and digitoxin for Sudlow’s site III were indicated. Competitive experiments have shown that palladium complex along with digitoxin have a common position on HSA at site III.

    Keywords: Competitive binding, Hsa, Fluorescence quenching, Oxalato palladium, Site marker
  • Reza Farahmand, Mehdi Ebrahimi * Pages 159-166
    α-Amylases catalyze the hydrolysis of starch and are widely used in various industries, especially in the food industry. Despite the high demand for the use of α-amylase in various industries, most α-amylases do not have the temperature and pH stabilities for use in industrial processes. Ionic liquids are broadly used as co-solvents to improve the activity, stability, and selectivity of enzymes. In this study, the effect of 1-Ethyl-3-methylimidazolium Acetate on the activity, temperature and pH optimum, thermal stability, and structure of an α-amylase from Aspergillus oryzae were investigated. The activity of α-amylase was decreased with increments in [EMIm][Ac] concentration. Vm of α-amylase in the presence of [EMIm][Ac] was increased while Km was decreased. Same as the absence of [EMIm][Ac], the optimal pH and temperature in the presence of [EMIm][Ac] were 7.5 and 45 °C, respectively. The inactivation rate of α-amylase at temperatures of 40, 50, and 60 °C in the presence of both 0.4 and 1 M [EMIm][Ac] is less than in its absence. Intrinsic fluorescence spectroscopy revealed structural changes in the presence of [EMIm][Ac]. These results indicate the improvements in the activity, optimum temperature, and thermal stability of α-amylase due to the structural changes induced with [EMIm][Ac].
    Keywords: Ionic liquids, [EMIm][Ac], Fluorescence spectroscopy
  • Zahra Shabaninejad, Maryam Nikkhah *, Masoud Nabavizadeh Pages 167-173
    Platinum-containing compounds are among organometallic compounds that are considered promising drug candidates for cancer therapy. They have an important role in the treatment of several solid tumors. The interactions of small molecule drugs with serum albumin affect the pharmacokinetics of these therapeutic agents. In this study, the binding characteristics of two novel platinum complexes, named C1 and C2, and bovine serum albumin (BSA) were investigated by fluorescence and UV-visible spectroscopies. The results showed that these compounds diminished the fluorescence emission intensity of BSA through ground state quenching mechanisms. The apparent binding constant (Kb) values for the association of these compounds and BSA were in the range of 103-104 M-1, showing their moderate affinity to BSA. The Kb and the numbers of binding sites of BSA-complexes were increased by the elevation of temperature indicating that binding pockets were more accessible to these compounds at higher temperatures. Thermodynamic parameters showed that hydrophobic interactions are involved in C1-BSA complex formation and the interaction of C2 with BSA is through van der Waals interactions and hydrogen bonds.
    Keywords: BSA, Platinum complexes, Fluorescence quenching, Thermodynamic parameter
  • Mojtaba Mortazavi, Abdolrasool Malekpour, Farzaneh Kargar, Saeid Gholamzadeh *, Reza Mohammadi, Zahra Mohammadi Pages 174-184
    Coronaviruses are enveloped single-stranded RNA genome viruses causing respiratory distress syndromes. The aim of this study was to compare codon usage, rare codon clusters and phylogenetic relations in orf1a polyprotein of COVID-19, SARS, and MERS coronaviruses. The frequency, number and fraction of 61 codons for each amino acid was evaluated in the structure of viral protein and the preferred codons were assessed using Gene Infinity website. The variations in codon usage bias were quantified by the ENC and CBI in the ACUA software. Finally, evolutionary relationship and phylogenetic analysis of Coronaviridae were studied using the MEGA 7 software. The GC3% of the cds was in the range from 15.668 to 16.534 and GC3 Skewness from 0.299 to 0.34. Analysis of codon usage for all of amino acid in SARS, MERS and COVID-19 showed considerable differences between three viruses. The close proximity of COVID-19 and SARS in the tree diagram represented a similarity in their gene sequence of orf1a polyprotein (pp1a). This phylogenetic analysis also indicated that COVID-19 and SARS had the near phylogenetic relation based on the nucleotide sequences of orf1a polyprotein in comparison to MERS. The findings of the present study revealed that the patterns of base compositions in COVID-19 are most likely the result of mutation pressure rather than that of natural selection, since at all codon positions its effects are present. In addition, analysis of base composition it was found that the cds of COVID-19 are rich in AT, which should be considered in designing new drugs.
    Keywords: Computational Biology, SARS, MERS, COVID-19