Molecular Biotechnology

Maryam Ahmadzadeh, Farzaneh Farshdari, Leila Nematollahi, Mahdi Behdani, Elham Mohit
Breast cancer is the second most commonly diagnosed cancer, worldwide. Human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer is correlated with poor prognosis. HER2-targeting monoclonal antibodies resulted in longer survival of HER2+ breast cancer. Single-chain variable fragment (scFv) demonstrates improved penetrability into tumors. Due to the presence of two disulfide bond, scFv expression in reducing bacterial cytoplasm may cause formation of inclusion bodies. Disulfide bond can be formed properly in cytoplasm of SHuffle® strain as it is trxB- , gor- , and overexpresses cytoplasmic DsbC chaperone...
November 5, 2019: Molecular Biotechnology
Rahul G Shelke, Latha Rangan
Pongamia pinnata (also called Millettia pinnata), a non-edible oil yielding tree, is well known for its multipurpose benefits and acts as a potential source for medicine and biodiesel preparation. Due to increase in demand for cultivation, understanding of genetic diversity is an important parameter for further breeding and cultivation programme. Transposable elements (TEs) are a major component of plant genome but still, their evolutionary significance in Pongamia remains unexplored. In view to understand the role of TEs in genome diversity, Pongamia unigenes were screened for the presence of TE cassettes...
October 31, 2019: Molecular Biotechnology
C Sunisha, H D Sowmya, T R Usharani, M Umesha, H R Gopalkrishna, Arvindkumar Saxena
Enhanced tolerance to wilt disease (Fusarium oxysporum f.sp. cubense) was achieved in banana variety Rasthali (AAB) by the transformation of embryogenic cells with two antimicrobial genes viz., Ace-AMP1 and pflp using Agrobacterium mediated transformation. The transgene copy numbers in stable transformants were confirmed by Southern analysis. The expression of stacked genes in the transgenic lines was validated by RT-PCR as well as Northern analysis. Bioassay using Foc race 1 in pot culture experiments demonstrated enhanced tolerance after 180 days of planting...
October 30, 2019: Molecular Biotechnology
Varsha Acharya, Hirak Jyoti Chakraborty, Ajaya Kumar Rout, Sucharita Balabantaray, Bijay Kumar Behera, Basanta Kumar Das
In recent years, large-scale mortalities are observed in tilapia due to infection with a novel orthomyxo-like virus named, tilapia lake virus (TiLV) which is marked to be a severe threat to universal tilapia industry. Currently, there are knowledge gaps relating to the antiviral peptide as well as there are no affordable vaccines or drugs available against TiLV yet. To understand the spreading of infection of TiLV in different organs of Oreochromis niloticus, RT-PCR analysis has been carried out. The gene segments of TiLV were retrieved from the NCBI database for computational biology analysis...
October 29, 2019: Molecular Biotechnology
Christos Papaneophytou
In this review, the basic concepts and applications of design of experiments (DoE) in recombinant protein biotechnology will be discussed. The production of recombinant proteins usually begins with the construction of an expression vector that is then introduced into a microbial host. The target protein is overexpressed in the host's cells and subsequently, it is isolated using a suitable purification method, its activity is assessed using a biological assay, while its crystallization is often required. Because each protein is unique and due to the complex interactions among the reagents in experiments, it is impossible that one set of reaction conditions would be optimal for all cases...
October 29, 2019: Molecular Biotechnology
Fang Nan, Junwei Jiang, Shenglu Wu, Yueqi Zhang, Jiarong Qiu, Beibei Qiao, Shan Li, Zhihong Xin
A novel carboxylesterase gene, named dlfae4, was discovered and sequenced from a soil metagenomic library. The dlfae4 gene was composed of 1017 base pairs encoding 338 amino acid residues with a predicted molecular mass of 37.2 kDa. DLFae4 exhibited strong hydrolytic activity towards methyl ferulate under optimum pH and temperature conditions (pH 8.6, 50 °C) and displayed remarkable thermostability, with residual activity as high as 50% after incubation for 3 h at 60 °C. A family VIII esterase DLFae4 was found to contain a typical serine residue within the S-X-X-K motif, which serves as a catalytic nucleophile in class C β-lactamases and family VIII esterases...
October 29, 2019: Molecular Biotechnology
Shuhong Lu, Xuesong Zhang, Kaiying Chen, Bingbin Xie, Dapeng Shan, Yulong Shen, Zhuo Li
The Hot Start polymerase chain reaction (Hot Start PCR) is designed to reduce off-target amplification by blocking DNA polymerase extension at room temperature until the desired temperature is reached. In this study, we investigated a new method of Hot Start PCR that uses a modified Escherichia coli Exonuclease III (EcoExoIIIM) by substituting residues in the DNA-binding pocket and catalytic center. The results showed that PCR amplification yield and specificity were significantly promoted by the addition of EcoExoIIIM...
October 22, 2019: Molecular Biotechnology
Zahra Farrokhi, Hassan Alizadeh, Houshang Alizadeh, Fariba Abooei Mehrizi
Broomrape is an obligate root-parasitic weed that acts as a competitive sink for host photoassimilates. Disruption of essential processes for growth of broomrape using host plant-mediated systemic signals can help to implement more specific and effective management plans of this parasite. Accordingly, we tested the possibility of transient silencing three involved genes (PaM6PR, PaCWI, and PaSUS1) in osmoregulation process of broomrape using syringe agroinfiltration of dsRNA constructs in tomato. The highest decrease in mRNA levels, enzyme activity, and amount of total reducing sugars was observed in Phelipanche aegyptiaca when grown on agroinfiltrated tomato plants by PaM6PR dsRNA construct than control...
September 28, 2019: Molecular Biotechnology
Dominika Durechova, Martin Jopcik, Miroslav Rajninec, Jana Moravcikova, Jana Libantova
In this study, a chitinase gene (DrChit) that plays a role in the carnivorous processes of Drosera rotundifolia L. was isolated from genomic DNA, linked to a double CaMV35S promoter and nos terminator in a pBinPlus plant binary vector, and used for Agrobacterium-mediated transformation of tobacco. RT-qPCR revealed that within 14 transgenic lines analysed in detail, 57% had DrChit transcript abundance comparable to or lower than level of a reference actin gene transcript. In contrast, the transgenic lines 9 and 14 exhibited 72 and 152 times higher expression level than actin...
September 25, 2019: Molecular Biotechnology
Gayathri Purushothaman, Vijay Thiruvenkatam
CD151 is an abundantly expressed eukaryotic transmembrane protein on the cell surface. It is involved in cell adhesion, angiogenesis and signal transduction as well in disease conditions such as cancer and viral infections. However, the molecular mechanism of CD151 activation is poorly understood due to the lack of structural information. By considering the difficulties in expressing the membrane protein in E. coli, herein we introduce the strategic design for the effective expression of recombinant CD151 protein in E...
September 20, 2019: Molecular Biotechnology
Kashmala Shehryar, Raham Sher Khan, Aneela Iqbal, Syeda Andaleeb Hussain, Sawera Imdad, Anam Bibi, Laila Hamayun, Ikuo Nakamura
Introduction of more than one gene into crop plants simultaneously or sequentially, called transgene stacking, has been a more effective strategy for conferring higher and durable insect and disease resistance in transgenic plants than single-gene technology. Transgenes can be stacked against one or more pathogens or for traits such as herbicide tolerance or anthocyanin pigmentation. Polygenic agronomic traits can be improved by multiple gene transformation. The most widely engineered stacked traits are insect resistance and herbicide tolerance as these traits may lead to lesser use of pesticides, higher yield, and efficient control of weeds...
September 19, 2019: Molecular Biotechnology
Rossella Di Stasi, Donatella Diana, Lucia De Rosa, Roberto Fattorusso, Luca D D'Andrea
Angiogenesis is a biological process finely tuned by a plethora of pro- and anti-angiogenic molecules, among which vascular endothelial growth factors (VEGFs). Their biological activity is expressed through the interaction with three cognate receptor tyrosine kinases, VEGFR1, 2, and 3. VEGFR2 is the primary regulator of angiogenesis. Ligand-induced VEGFR2 dimerization and activation depend on direct ligand binding to extracellular domains 2 and 3 of receptor and in the establishment of interactions between proximal membrane domains...
September 17, 2019: Molecular Biotechnology
Lu Bian, Zhen Zhang, Rong-Xing Tang, Wei Shen, Li-Xin Ma
Methyl parathion hydrolase (MPH) plays an important role in degrading a range of organophosphorus compounds. In order to display MPH on the cell surface of Escherichia coli strain RosettaBlue™, the Flavin-based fluorescent protein EcFbFP was severed as an auto-anchoring matrix. With net negative charges of EcFbFP supplying the driving forces, fusion protein MPH-EcFbFP through a two-step auto-surface display process was finally verified by (a) inner membrane translocation and (b) anchoring at outer membrane...
September 5, 2019: Molecular Biotechnology
Abdullahi Adekilekun Jimoh, Johnson Lin
Phosphopantetheinyl transferases are of tremendous enthusiasm inferable from their fundamental parts in activating polyketide, fatty acid, and non-ribosomal peptide synthetase enzymes and additionally an increasing number of biotechnological applications. The present study reports the identification of sfp gene from the Paenibacillus sp. D9, which encompasses 693 bp encoding a 230-amino acid protein with a molecular weight of 25.3 kDa. The amino acid sequence Paenibacillus sp. D9 Sfp revealed more than 90% sequence identity to other Sfp proteins from other Paenibacillus...
September 3, 2019: Molecular Biotechnology
Liu Chen, Bin Yu, Jonggang Hua, Zheng Ni, Weicheng Ye, Tao Yun, Cun Zhang
In our previous study, a recombinant duck enteritis virus (DEV) delivering codon-optimized E gene (named as E-ch) of duck Tembusu virus (DTMUV) optimized referring to chicken's codon bias has been obtained based on the infectious bacterial artificial chromosome (BAC) clone of duck enteritis virus vaccine strain pDEV-EF1, but the expression level of E-ch in recombinant virus rDEV-E-ch-infected cells was very low. To optimize DTMUV E gene expression delivered by the vectored DEV, different forms of E gene (collectively called EG) including origin E gene (E-ori), truncated E451-ori gene, codon-optimized E-dk gene optimized referring to duck's codon bias, as well as the truncated E451-ch and E451-dk, Etpa-ori and Etpa-451-ori, which contain prefixing chick TPA signal peptide genes, were cloned into transfer vector pEP-BGH-end, and several recombinant plasmids pEP-BGH-EG were constructed...
September 3, 2019: Molecular Biotechnology
Doddy Irawan Setyo Utomo, Ichikawa Hirono, Tatsuya Kato, Enoch Y Park
To explore virus-like particles formation of dengue virus serotype type 2 (DENV-2) structural proteins of, C, prM, E were expressed in silkworm larvae using recombinant Bombyx mori nucleopolyhedroviruses (BmNPV). Each recombinant BmNPV bacmid coding the 2C-prM-E polypeptide and E protein fused with the signal peptide of bombyxin from B. mori was injected into silkworm larvae. The expressed proteins were collected from hemolymph and fat body, and purified using affinity chromatography. E protein was observed at 55 kDa...
August 31, 2019: Molecular Biotechnology
Rose H Reader, Robert G Workman, Ben C Maddison, Kevin C Gough
Phage display antibody libraries have proven an invaluable resource for the isolation of diagnostic and potentially therapeutic antibodies, the latter usually being antibody fragments converted into IgG formats. Recent advances in the production of highly diverse and functional antibody libraries are considered here, including for Fabs, scFvs and nanobodies. These advances include codon optimisation during generation of CDR diversity, improved display levels using novel signal sequences, molecular chaperones and isomerases and the use of highly stable scaffolds with relatively high expression levels...
August 29, 2019: Molecular Biotechnology
Anna Mądry, Agnieszka Jendroszek, Grzegorz Dubin, Benedykt Wladyka
Lysostaphin is a staphylolytic protein of growing interest from biotechnological and pharmaceutical industry due to its potential use in preventing and combating staphylococcal infections. Here, we describe an optimized method for production of lysostaphin in an inductionless system utilizing constitutive promoter from staphylococcal toxin-antitoxin system PemIK-Sa1. We investigated the influence of ribosome-binding site sequence, Escherichia coli producer strain and growth media on yield and kinetics of recombinant protein production...
August 24, 2019: Molecular Biotechnology
Lu-Zhou Chen, Chu-Qi Shi, Feng-Xin Yin, Feng-Shan Wang, Ju-Zheng Sheng
Glycosaminoglycans (GAGs) and their low-molecular weight derivates have received considerable interest in terms of their potential clinical applications, and display a wide variety of pharmacological and pharmacokinetic properties. Structurally distinct GAG chains can be prepared by enzymatic depolymerization. A variety of bacterial chondroitin sulfate (CS) lyases have been identified, and have been widely used as catalysts in this process. Here, we identified a putative chondroitin AC exolyase gene, AschnAC, from an Arthrobacter sp...
August 23, 2019: Molecular Biotechnology
Dishant Goyal, Krishan Kumar, Maria S J Centeno, Abhijeet Thakur, Virgínia M R Pires, Pedro Bule, Carlos M G A Fontes, Arun Goyal
The cellulosomal enzyme, RfGH51/2 , of Ruminococcus flavefaciens contains an N-terminal module, a family 5 glycoside hydrolase GH5_4 with a putative endoglucanase activity, while C-terminal domain is a putative endo-mannanase (GH5_7). The two putative catalytic modules are separated by family 80 carbohydrate binding module (CBM80) having wide ligand specificity. The putative endo-mannanase module, GH5_7 (RfGH5_7), was cloned, expressed in Escherichia coli BL-21(DE3) cells and purified. SDS-PAGE analysis of purified RfGH5_7 showed molecular size ~ 35 kDa...
August 21, 2019: Molecular Biotechnology
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