共查询到20条相似文献，搜索用时 78 毫秒
1.
Hua Deng Prashanta Dutta Jin Liu 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(9):2104-2111
Background
Receptor dependent clathrin-mediated endocytosis (CME) is one of the most important endocytic pathways for the internalization of bioparticles into cells. During CME, the ligand-receptor interactions, development of clathrin-coated pit (CCP) and membrane evolution all act together to drive the internalization of bioparticles. In this work, we develop a stochastic computational model to investigate the CME based on the Metropolis Monte Carlo simulations.Methods
The model is based on the combination of a stochastic particle binding model with a membrane model. The energetic costs of membrane bending, CCP formation and ligand-receptor interactions are systematically linked together.Results
We implement our model to investigate the effects of particle size, ligand density and membrane stiffness on the overall process of CME from the drug delivery perspectives. Consistent with some experiments, our results show that the intermediate particle size and ligand density favor the particle internalization. Moreover, our results show that it is easier for a particle to enter a cell with softer membrane.Conclusions
The model presented here is able to provide mechanistic insights into CME and can be readily modified to include other important factors, such as actins. The predictions from the model will aid in the therapeutic design of intracellular/transcellular drug delivery and antiviral interventions. 相似文献2.
Yuta Murakami Koichi Takahashi Kyoka Hoshi Hiromi Ito Mayumi Kanno Kiyoshi Saito Kenneth Nollet Yoshiki Yamaguchi Masakazu Miyajima Hajime Arai Yasuhiro Hashimoto Tatsuo Mima 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(8):1835-1842
Background
Spontaneous intracranial hypotension (SIH) is caused by cerebrospinal fluid (CSF) leakage. Definitive diagnosis can be difficult by clinical examinations and imaging studies.Methods
SIH was diagnosed with the following criteria: (i) evidence of CSF leakage by cranial magnetic resonance imaging (MRI) findings of intracranial hypotension and/or low CSF opening pressure; (ii) no recent history of dural puncture. We quantified CSF proteins by ELISA or Western blotting.Results
Comparing with non-SIH patients, SIH patients showed significant increase of brain-derived CSF glycoproteins such as lipocalin-type prostaglandin D synthase (L-PGDS), soluble protein fragments generated from amyloid precursor protein (sAPP) and “brain-type” transferrin (Tf). Serum-derived proteins such as albumin, immunoglobulin G, and serum Tf were also increased. A combination of L-PGDS and brain-type Tf differentiated SIH from non-SIH with sensitivity 94.7% and specificity 72.6%.Conclusion
L-PGDS and brain-type Tf can be biomarkers for diagnosing SIH.General significance
L-PGDS and brain-type Tf biosynthesized in the brain appears to be markers for abnormal metabolism of CSF. 相似文献3.
Peng Zhao Bo Chen Lushen Li Hao Wu Yan Li Baxter Shaneen Xi Zhan Ning Gu 《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(2):502-510
Background
Magnetic nanoparticles (MNPs) have been widely used in biomedical applications. Proper control of the duration of MNPs in circulation promises to improve further their applications, in particularly drug delivery. It is known that the uptake of tissue-associated MNPs is mainly carried out by macrophages. Yet, the molecular mechanism to control MNPs internalization in macrophages remains to be elusive. Missing-in-metastasis (MIM) is a scaffolding protein that is highly expressed in macrophages and regulates receptor-mediated endocytosis. We hypothesize that uptake of MNPs may also involve the function of MIM.Methods
We investigated the effect of MIM expression on the intracellular trafficking of MNPs by transmission electronic microscopy, flow cytometry, o-phenanthroline photometric analysis, Perl's staining, immunofluorescence microscopy and co-immunoprecipitation. To explore the molecular events in MIM-mediated MNPs uptake, we examined the effect of MNPs on the interaction of MIM with clathrin, Rab5 and Rab7.Results
Uptake of MNPs was significantly enhanced in cells overexpressing MIM. Upon exposure to MNPs, MIM was associated with clathrin light chain in endocytic vesicles and Rab7, a protein that regulates late endosomes. However, MNPs caused dissociation of MIM with Rab5, an early endosome-associated protein.Conclusions
MIM regulates internalization of MNPs via promoting their trafficking from plasma membrane to late endosomes.General significance
Our data unveiled a novel pathway which MNPs internalization and intracellular trafficking in macrophages. This new pathway may allow us to control the uptake of MNPs within cells by targeting MIM, thereby improving their medical applications. 相似文献4.
Seong-Cheol Park Il Ryong Kim Jin-Young Kim Yongjae Lee Eun-Ji Kim Ji Hyun Jung Young Jun Jung Mi-Kyeong Jang Jung Ro Lee 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(12):2545-2554
Background
It remains an open question whether plant phloem sap proteins are functionally involved in plant defense mechanisms.Methods
The antifungal effects of two profilin proteins from Arabidopsis thaliana, AtPFN1 and AtPFN2, were tested against 11 molds and 4 yeast fungal strains. Fluorescence profiling, biophysical, and biochemical analyses were employed to investigate their antifungal mechanism.Results
Recombinant AtPFN1 and AtPFN2 proteins, expressed in Escherichia coli, inhibited the cell growth of various pathogenic fungal strains at concentrations ranging from 10 to 160?μg/mL. The proteins showed significant intracellular accumulation and cell-binding affinity for fungal cells. Interestingly, the AtPFN proteins could penetrate the fungal cell wall and membrane and act as inhibitors of fungal growth via generation of cellular reactive oxygen species and mitochondrial superoxide. This triggered the AtPFN variant-induced cell apoptosis, resulting in morphological changes in the cells.Conclusion
PFNs may play a critical role as antifungal proteins in the Arabidopsis defense system against fungal pathogen attacks.General significance
The present study indicates that two profilin proteins, AtPFN1 and AtPFN2, can act as natural antimicrobial agents in the plant defense system. 相似文献5.
6.
Viacheslav V. Senichkin Gelina S. Kopeina Eugeniia A. Prokhorova Alexey V. Zamaraev Inna N. Lavrik Boris Zhivotovsky 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(3):557-566
Background
The development of approaches that increase therapeutic effects of anti-cancer drugs is one of the most important tasks of oncology. Caloric restriction in vivo or serum deprivation (SD) in vitro has been shown to be an effective tool for sensitizing cancer cells to chemotherapeutic drugs. However, the detailed mechanisms underlying the enhancement of apoptosis in cancer cells by SD remain to be elucidated.Methods
Flow cytometry, caspase activity assay and western blotting were used for cell death rate evaluation. Western blotting, gel-filtration, siRNA approach and qRT-PCR were used to elucidate the mechanism underlying cell death potentiation upon SD.Results
We demonstrated that SD sensitizes cancer cells to treatment with chemotherapeutic agent cisplatin. This effect is independent on activation of caspases-2 and -8, apical caspases triggering apoptosis in response to genotoxic stress. SD potentiates cell death via downregulation of the anti-apoptotic protein Mcl-1. In fact, SD reduces the Mcl-1 mRNA level, which consequently decreases the Mcl-1 protein level and renders cells more susceptible to apoptosis induction via the formation of apoptosome.Conclusions
Mcl-1 protein is an important regulator of sensitivity of cancer cells to apoptotic stimuli upon SD.General significance
This study identifies Mcl-1 as a new target for the sensitization of human cancer cells to cell death by SD, which is of great significance for the development of efficient anti-cancer therapies. 相似文献7.
Natalia V. Dolgova Susan Nehzati Sanjukta Choudhury Tracy C. MacDonald Nathan R. Regnier Andrew M. Crawford Olena Ponomarenko Graham N. George Ingrid J. Pickering 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(11):2383-2392
Background
Selenium is an essential element with a rich and varied chemistry in living organisms. It plays a variety of important roles ranging from being essential in enzymes that are critical for redox homeostasis to acting as a deterrent for herbivory in hyperaccumulating plants. Despite its importance there are many open questions, especially related to its chemistry in situ within living organisms.Scope of review
This review discusses X-ray spectroscopy and imaging of selenium in biological samples, with an emphasis on the methods, and in particular the techniques of X-ray absorption spectroscopy (XAS) and X-ray fluorescence imaging (XFI). We discuss the experimental methods and capabilities of XAS and XFI, and review their advantages and their limitations. A perspective on future possibilities and next-generation of experiments is also provided.Major conclusions
XAS and XFI provide powerful probes of selenium chemistry, together with unique in situ capabilities. The opportunities and capabilities of the next generation of advanced X-ray spectroscopy experiments are particularly exciting.General significance
XAS and XFI provide versatile tools that are generally applicable to any element with a convenient X-ray absorption edge, suitable for investigating complex systems essentially without pre-treatment. 相似文献8.
Wataru Yoshida Mizuki Terasaka Saowalak Laddachote Isao Karube 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(9):1933-1937
Background
DNA methylation at the 5-position of cytosine is an epigenetic modification of CpG dinucleotides. In addition to CpG methylation, the G-quadruplex (G4) structure has been reported as a regulator of gene expression. The identification of G4 forming sequences in CpG islands suggests an involvement of CpG-methylated G4 structures in biological processes; however, few reports have addressed the effects of CpG methylation on G4 structure.Methods
The thermostability of a methylated, 21-mer G4 structure located on the vascular endothelial growth factor (VEGF) gene promoter containing four CpG sites (C1, C6, C11, and C17) were investigated using circular dichroism (CD) spectral analysis.Results
CD melting analysis revealed that VEGF G4 was stabilized by a single CpG methylation on C11 in the presence of Na+ and Mg2+. However, either C1 or C11 methylation enhanced VEGF G4 thermal stability in the presence of K+.Conclusions
Single CpG methylation appears to enhance VEGF G4 thermostability in a manner dependent on both the CpG methylation site and cation type.General significance
These results are expected to contribute to the elucidation of the roles of CpG methylation-stabilized G4 structures in biological processes. 相似文献9.
Christopher E. Ramsden Marie Hennebelle Susanne Schuster Gregory S. Keyes Casey D. Johnson Irina A. Kirpich Jeff E. Dahlen Mark S. Horowitz Daisy Zamora Ariel E. Feldstein Craig J. McClain Beverly S. Muhlhausler Maria Makrides Robert A. Gibson Ameer Y. Taha 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2018,1863(10):1206-1213
Background
Linoleic acid (LA) is abundant in modern industrialized diets. Oxidized LA metabolites (OXLAMs) and reactive aldehydes, such as 4-hydroxy-2-nonenal (4-HNE), are present in heated vegetable oils and can be endogenously synthesized following consumption of dietary LA. OXLAMs have been implicated in cerebellar degeneration in chicks; 4-HNE is linked to neurodegenerative conditions in mammals. It unknown whether increasing dietary LA or OXLAMs alters the levels of oxidized fatty acids (oxylipins), precursor fatty acids, or 4-HNE in mammalian brain.Objectives
To determine the effects of increases in dietary OXLAMs and dietary LA, on levels of fatty acids, oxylipins, and 4-HNE in mouse brain tissues.Methods
Mice (n?=?8 per group) were fed one of three controlled diets for 8?weeks: (1) a low LA diet, (2) a high LA diet, or (3) the low LA diet with added OXLAMs. Brain fatty acids, oxylipins, and 4-HNE were quantified in mouse cerebellum and cerebral cortex by gas chromatography-flame ionization detection, liquid chromatography-tandem mass spectrometry, and immunoblot, respectively.Results
Increasing dietary LA significantly increased omega-6 fatty acids, decreased omega-3 fatty acids, and increased OXLAMs in brain. Dietary OXLAMs had minimal effect on oxidized lipids but did decrease both omega-6 and omega-3 fatty acids. Neither dietary LA nor OXLAMs altered 4-HNE levels.Conclusion
Brain fatty acids are modulated by both dietary LA and OXLAMs, while brain OXLAMs are regulated by endogenous synthesis from LA, rather than incorporation of preformed OXLAMs. 相似文献10.
Nikolay A. Barinov Irina I. Vlasova Alexey V. Sokolov Valeria A. Kostevich Evgeniy V. Dubrovin Dmitry V. Klinov 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(12):2862-2868
Background
Metalloproteins myeloperoxidase (MPO), ceruloplasmin (CP) and lactoferrin (LF) play an important role in regulation of inflammation and oxidative stress in vertebrates. It was previously shown that these proteins may work synergetically as antimicrobial and anti-inflammatory agents by forming complexes, such as MPO-CP and LF-CP. However, interaction of metalloprotein molecules with each other has never been characterized at a single-molecule level.Methods
In this study, the pairwise interactions of MPO, CP and LF molecules were investigated at a single-molecule level using high-resolution atomic force microscopy (AFM). Highly oriented pyrolytic graphite surface (HOPG) modified with oligoglycine-hydrocarbon graphite modifier (GM) was used as a substrate for protein deposition.Results
The procedure for reliable AFM investigation of metalloproteins and their complexes has been developed. Using this procedure, we have visualized, for the first time, single MPO, CP and LF molecules, characterized the morphology of MPO-CP and LF-CP complexes and confirmed the absence of direct contacts between MPO and LF molecules. Moreover, we have revealed the novel chainlike shape of MPO-CP conjugates.Conclusions
GM-HOPG was shown to be a convenient substrate for AFM investigation of metalloproteins and their complexes. Direct AFM visualization of MPO-CP and LF-CP complexes, on the one hand, complements previous data obtained from the “bulk techniques” and, on the other hand, provides new insight into the ultrastructure of MPO-CP complexes.General significance
The obtained results contribute to the better understanding of regulation of inflammation and oxidation stress mediated by collaborative action of the metalloproteins such as MPO, CP and LF. 相似文献11.
Meenakshi Sharma Dinesh Kumar Krishna Mohan Poluri 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(4):924-935
Background
Characterization of partially collapsed protein conformations at atomic level is a daunting task due to their inherent flexibility and conformational heterogeneity. T7 bacteriophage endolysin (T7L) is a single-domain amidase that facilitates the lysis of Gram-negative bacteria. T7L exhibits a pH-dependent structural transition from native state to partially folded (PF) conformation. In the pH range 5–3, T7L PF states display differential ANS binding characteristics.Methods
CD, fluorescence, NMR spectroscopy and lysis assays were used to investigate the structure-stability- dynamics relationships of T7L PF conformations.Results
Structural studies indicated a partial loss of secondary/tertiary structures compared to its native state. The loss in the tertiary structure and the hydrophobic core opening increases upon decrease of pH from 5 to 3. Thermal denaturation experiments delineated that the pH?5 conformation is thermally irreversible in contrast to pH?3, depicting that hydrophobic core opening is essential for thermal reversibility. Further, urea dependent unfolding features of PF state at pH?5 and 4 evidenced for a collapsed conformation at intermediate urea concentrations. Residue level studies revealed that α1-helix and β3-β4 segment of T7L are the major contributors for such a structural collapse and inherent dynamics.Conclusions
The results suggested that the low pH PF states of T7L are heterogeneous and exhibits differential structural, unfolding, thermal reversibility, and dynamic features.General significance
Unraveling the structure-stability characteristics of different endolysin conformations is essential for designing novel chimeric and engineered phage endolysins as broadband antimicrobial agents over a varied pH range. 相似文献12.
Sara Lago Matteo Nadai Monica Rossetto Sara N. Richter 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(6):1276-1282
Background
G-quadruplexes (G4s) are nucleic acids secondary structures formed in guanine-rich sequences. Anti-G4 antibodies represent a tool for the direct investigation of G4s in cells. Surface Plasmon Resonance (SPR) is a highly sensitive technology, suitable for assessing the affinity between biomolecules. We here aimed at improving the orientation of an anti-G4 antibody on the SPR sensor chip to optimize detection of binding antigens.Methods
SPR was employed to characterize the anti-G4 antibody interaction with G4 and non-G4 oligonucleotides. Dextran-functionalized sensor chips were used both in covalent coupling and capturing procedures.Results
The use of two leading molecule for orienting the antibody of interest allowed to improve its activity from completely non-functional to 65% active. The specificity of the anti-G4 antobody for G4 structures could thus be assessed with high sensitivity and reliability.Conclusions
Optimization of the immobilization protocol for SPR biosensing, allowed us to determine the anti-G4 antibody affinity and specificity for G4 antigens with higher sensitivity with respect to other in vitro assays such as ELISA. Anti-G4 antibody specificity is a fundamental assumption for the future utilization of this kind of antibodies for monitoring G4s directly in cells.General significance
The heterogeneous orientation of amine-coupling immobilized ligands is a general problem that often leads to partial or complete inactivation of the molecules. Here we describe a new strategy for improving ligand orientation: driving it from two sides. This principle can be virtually applied to every molecule that loses its activity or is poorly immobilized after standard coupling to the SPR chip surface. 相似文献13.
Veronica Esposito Annapina Russo Valentina Vellecco Mariarosaria Bucci Giulia Russo Luciano Mayol Antonella Virgilio Aldo Galeone 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(12):2645-2650
Background
Although the thrombin binding aptamer (TBA) is endowed with both anticoagulant and antiproliferative properties, it is possible to reduce the first and enhance the second one by suitable chemical modifications.Methods
Two oligonucleotides (TBA353 and TBA535) based on the TBA sequence (GGTTGGTGTGGTTGG) and containing inversion of polarity sites have been investigated by CD, UV and electrophoretic techniques for their ability to form G-quadruplex structures. Furthermore, their anticoagulant (PT assay), antiproliferative (MTT assay) and anti-motility (wound healing assay) properties against Calu-6 cells have been tested and compared with TBA.Results
CD, UV and electrophoresis data indicate that both ODNs are able to form G-quadruplex structures. Particularly, results suggest that TBA535 adopts a G-quadruplex structure characterized by a loop arrangement different from that of TBA. Both TBA analogues drop the anticoagulant activity. However, TBA535 is endowed with a significant antiproliferative activity against lung cancer Calu-6 cells. Importantly, both TBA and TBA535 possess a remarkable anti-motility property against the same cell line.Conclusions
Both TBA analogues TBA353 and TBA535 are able to form G-quadruplex structures with no anticoagulant activity. However only TBA535 is endowed with noteworthy antiproliferative and anti-motility properties against lung cancer Calu-6 cells.General significance
The switching from the anticoagulant to antiproliferative property can be obtained also in TBA derivatives not adopting the “chair-like” G-quadruplex structure typical of TBA. Furthermore, results have highlighted an unprecedented anti-cell-motility property of TBA and TBA535 reinforcing the potential of these ODNs as anticancer drugs. 相似文献14.
Teppei Ikeya David Ban Donghan Lee Yutaka Ito Koichi Kato Christian Griesinger 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(2):287-306
Background
To understand the mechanisms related to the ‘dynamical ordering’ of macromolecules and biological systems, it is crucial to monitor, in detail, molecular interactions and their dynamics across multiple timescales. Solution nuclear magnetic resonance (NMR) spectroscopy is an ideal tool that can investigate biophysical events at the atomic level, in near-physiological buffer solutions, or even inside cells.Scope of review
In the past several decades, progress in solution NMR has significantly contributed to the elucidation of three-dimensional structures, the understanding of conformational motions, and the underlying thermodynamic and kinetic properties of biomacromolecules. This review discusses recent methodological development of NMR, their applications and some of the remaining challenges.Major conclusions
Although a major drawback of NMR is its difficulty in studying the dynamical ordering of larger biomolecular systems, current technologies have achieved considerable success in the structural analysis of substantially large proteins and biomolecular complexes over 1 MDa and have characterised a wide range of timescales across which biomolecular motion exists. While NMR is well suited to obtain local structure information in detail, it contributes valuable and unique information within hybrid approaches that combine complementary methodologies, including solution scattering and microscopic techniques.General significance
For living systems, the dynamic assembly and disassembly of macromolecular complexes is of utmost importance for cellular homeostasis and, if dysregulated, implied in human disease. It is thus instructive for the advancement of the study of the dynamical ordering to discuss the potential possibilities of solution NMR spectroscopy and its applications. This article is part of a Special Issue entitled “Biophysical Exploration of Dynamical Ordering of Biomolecular Systems” edited by Dr. Koichi Kato. 相似文献15.
Sumangala Shetty Paul R. Copeland 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(11):2506-2510
Background
Selenoprotein synthesis requires the reinterpretation of a UGA stop codon as one that encodes selenocysteine (Sec), a process that requires a set of dedicated translation factors. Among the mammalian selenoproteins, Selenoprotein P (SELENOP) is unique as it contains a selenocysteine-rich domain that requires multiple Sec incorporation events.Scope of review
In this review we elaborate on new data and current models that provide insight into how SELENOP is made.Major conclusions
SELENOP synthesis requires a specific set of factors and conditions.General significance
As the key protein required for proper selenium distribution, SELENOP stands out as a lynchpin selenoprotein that is essential for male fertility, proper neurologic function and selenium metabolism. 相似文献16.
D. Bellavia L. Raimondi V. Costa A. De Luca V. Carina M. Maglio M. Fini R. Alessandro G. Giavaresi 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(9):1893-1901
Background
Exosomes are nanovesicles actively secreted by potentially all cell types, including tumour cells, with the primary role of extracellular systemic communication mediators, both at autocrine and paracrine levels, at short and long distances. Recently, different studies have used exosomes as a delivery system for a plethora of different molecules, such as drugs, microRNAs and proteins. This has been made possible thanks to the simplicity in exosomes engineering, their great stability and versatility for applications in oncology as well as in regenerative medicine.Scope of review
The aim of this review is to provide information on the state-of-the-art and possible applications of engineered exosomes, both for cargo and specific cell-targeting, in different pathologies related to the musculoskeletal system.Major conclusions
The use of exosomes as therapeutic agents is rapidly evolving, different studies explore drug delivery with exosomes using different molecules, showing an enormous potential in various research fields such as oncology and regenerative medicine.General significance
However, despite the significant progress made by the different studies carried out, currently, the use of exosomes is not a therapeutic reality for the considerable difficulties to overcome. 相似文献17.
Simin Feng Zhuqing Dai Anna B. Liu Jinbao Huang Nihal Narsipur Grace Guo Bo Kong Kenneth Reuhl Wenyun Lu Zisheng Luo Chung S. Yang 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2018,1863(10):1274-1284
Objective
To investigate and compare the effects of two common dietary phytosterols, stigmasterol and β-sitosterol, in altering lipid metabolism and attenuating nonalcoholic fatty liver disease (NAFLD).Methods
Stigmasterol and β-sitosterol were administered to mice at 0.4% in a high-fat western-style diet (HFWD) for 17?weeks.Results
Stigmasterol and β-sitosterol significantly ameliorated HFWD-induced fatty liver and metabolic abnormalities, including elevated levels of hepatic total lipids, triacylglycerols, cholesterol and liver histopathology. Both phytosterols decreased the levels of intestinal bile acids, accompanied by markedly increased fecal lipid levels. In addition, they altered the expression of genes involved in lipid metabolism. β-Sitosterol was less effective in affecting most of these parameters. Lipidomic analysis of liver and serum samples showed that stigmasterol prevented the HFWD-induced elevation of some di- and triacylglycerol species and lowering of some phospholipid species. Stigmasterol also decreased serum levels of ceramides.Conclusion
Stigmasterol and β-sitosterol, at a dose corresponding to that suggested for humans by the FDA for lowering cholesterol levels, are shown to alleviate HFWD-induced NAFLD. Stigmasterol was more effective than β-sitosterol, possibly because of its suppression of hepatic lipogenic gene expression and modulation of circulating ceramide levels. 相似文献18.
19.
Rafal Krela Elzbieta Poreba Martyna Weglewska Tomasz Skrzypczak Krzysztof Lesniewicz 《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(3):521-527
Background
During standard gene cloning, the recombinant protein appearing in bacteria as the result of expression leakage very often inhibits cell proliferation leading to blocking of the cloning procedure. Although different approaches can reduce transgene basal expression, the recombinant proteins, which even in trace amounts inhibit bacterial growth, can completely prevent the cloning process.Methods
Working to solve the problem of DNase II-like cDNA cloning, we developed a novel cloning approach. The method is based on separate cloning of the 5′ and 3′ fragments of target cDNA into a vector in such a way that the short Multiple Cloning Site insertion remaining between both fragments changes the reading frame and prevents translation of mRNA arising as a result of promoter leakage. Subsequently, to get the vector with full, uninterrupted Open Reading Frame, the Multiple Cloning Site insertion is removed by in vitro restriction/ligation reactions, utilizing the unique restriction site present in native cDNA.Results
Using this designed method, we cloned a coding sequence of AcDNase II that is extremely toxic for bacteria cells. Then, we demonstrated the usefulness of the construct prepared in this way for overexpression of AcDNase II in eukaryotic cells.Conclusions
The designed method allows cloning of toxic protein coding sequences that cannot be cloned by standard methods.General significance
Cloning of cDNAs encoding toxic proteins is still a troublesome problem that hinders the progress of numerous studies. The method described here is a convenient solution to cloning problems that are common in research on toxic proteins. 相似文献20.
Irena Trbojević-Akmačić Frano Vučković Marija Vilaj Andrea Skelin Lennart C. Karssen Jasminka Krištić Julija Jurić Ana Momčilović Jelena Šimunović Massimo Mangino Manuela De Gregori Maurizio Marchesini Concetta Dagostino Jerko Štambuk Mislav Novokmet Richard Rauck Yurii S. Aulchenko Dragan Primorac Gordan Lauc 《Biochimica et Biophysica Acta (BBA)/General Subjects》2018,1862(10):2124-2133