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Cloning, Expression Analysis and Enzyme Activity Assays of the α-Carbonic Anhydrase Gene from Chlamydomonas sp. ICE-L
Springer Science and Business Media LLC - Tập 60 - Trang 21-30 - 2017
Changfeng Qu, Yingying He, Zhou Zheng, Meiling An, Lulu Li, Xixi Wang, Xiaodong He, Yibin Wang, Fangming Liu, Jinlai Miao
The α-carbonic anhydrase (α-CA) is a zinc ion-containing enzyme that catalyzes the hydration of carbon dioxide. In this paper, a full-length α-CA gene was cloned from Chlamydomonas sp. ICE-L using RT-PCR and RACE-PCR for bioinformatic analysis. The α-CA open reading frame obtained by PCR was cloned into a vector and transformed into Escherichia coli to generate α-CA-producing bacteria. The α-CA was highly expressed upon induction with isopropyl-β-d-thiogalactoside (IPTG) at a final concentration of 0.8 mM. A single band with a molecular weight of approximate 40 kDa expressed in the recombinant E. coli strain harboring the α-CA vector was observed in SDS-PAGE analysis. The carbon dioxide hydration activity and esterase activity of α-CA expressed by the recombinant strain were 0.404 U/mg and 0.319 U, respectively. In addition, three conditions, temperature, salinity and UVB radiation exposure, were selected to analyze α-CA transcription levels by qRT-PCR. The results suggested UVB exposure increased the expression of relative mRNA; meanwhile, the α-CA mRNA expression was rapidly induced by temperature and salinity stress, indicating that Chlamydomonas sp. ICE-L might modulate the α-CA mRNA expression to adapt to the extreme environments.
An Alternative Approach in Gateway® Cloning when the Bacterial Antibiotic Selection Cassettes of the Entry Clone and Destination Vector are the Same
Springer Science and Business Media LLC - Tập 54 - Trang 133-140 - 2012
Kamal Kumar, Saurabh Yadav, Savithri Purayannur, Praveen Kumar Verma
The Gateway® recombination technology has revolutionized the method of gene cloning for functional analyses and high-throughput ORFeome projects. In general, Gateway cloning is highly efficient because after LR recombination and bacterial transformation, only cells containing the recombinant destination clone are selected on an antibiotic selection plate. However, when the antibiotic resistance gene for bacterial selection is the same in the entry and destination vectors, the direct selection of recombinant destination clones on an antibiotic plate is difficult. Here, we demonstrate an efficient and comprehensive approach to obtain positive destination clones directly on an antibiotic selection plate in this situation. The strategy involves polymerase chain reaction (PCR)-mediated amplification of the entry clone using entry vector-specific primers that bind outside the attL sequences and the subsequent use of this purified PCR product for LR recombination with the destination vector. Our results suggest that cloning of linear DNA fragments into circular destination vectors through LR recombination is an efficient method for inserts up to 7 kb in size. Using this approach, the yield of colony PCR positive destination clones was 100 % for genes of various sizes tested in our experiments.
Protein Misfolding, Aggregation, and Degradation in Disease<SUP>
Springer Science and Business Media LLC - Tập 31 Số 2 - Trang 141-150
Niels Gregersen, Lars Bolund, Peter Bross
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Functional Characterization of Recombinant Bromelain of Ananas comosus Expressed in a Prokaryotic System
Springer Science and Business Media LLC - Tập 56 - Trang 166-174 - 2013
Susan George, Salini Bhasker, Harish Madhav, Archana Nair, Mohankumar Chinnamma
Bromelain (BRM) is a defense protein present in the fruit and stem of pineapple (Ananas comosus) and it is grouped as a cysteine protease enzyme with diversified medicinal uses. Based on its therapeutic applications, bromelain has got sufficient attention in pharmaceutical industries. In the present study, the full coding gene of bromelain in pineapple stem (1,093 bp) was amplified by RT-PCR. The PCR product was cloned, sequenced, and characterized. The sequence analysis of the gene revealed the single nucleotide polymorphism and its phylogenetic relatedness. The peptide sequence deduced from the gene showed the amino acid variations, physicochemical properties and secondary and tertiary structural features of the protein. The full BRM gene was transformed to prokaryotic vector pET32b and expressed in Escherichia coli BL21 DE3pLysS host cells successfully. The identity of the recombinant bromelain (rBRM) protein was confirmed by Western blot analysis using anti-BRM-rabbit IgG antibody. The activity of recombinant bromelain compared with purified native bromelain was determined by protease assay. The inhibitory effect of rBRM compared with native BRM in the growth of Gram-positive and Gram-negative strains of Streptococcus agalactiae and Escherichia coli O111 was evident from the antibacterial sensitivity test. To the best of our knowledge, this is the first report showing the bactericidal property of rBRM expressed in a prokaryotic system.
Reverse Transcriptase-Polymerase Chain Reaction to Evaluate Human Cytomegalovirus Lytic Gene Expression
Springer Science and Business Media LLC - Tập 40 - Trang 144-150 - 2008
Massimiliano Bergallo, Cristina Costa, Maria Elena Terlizzi, Samuela Margio, Francesca Sidoti, Sara Astegiano, Franca Sinesi, Rossana Cavallo
This paper describes the development of four Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assays devised to evaluate lytic (UL123, immediate-early; UL54, early; UL65, late; and UL99, true late) human cytomegalovirus (HCMV) transcripts. Subsequently, the assays have been validated evaluating the HCMV lytic gene expression in 28 samples (peripheral blood leukocytes, PBLs) from 14 renal transplant recipients. Although the assessment of HCMV transcriptional profile could be useful to evaluate viral reactivation state, further studies on dynamics of viral transcripts in different clinical settings are needed to determine the role of transcriptional profiling in virological monitoring and therapeutic management in immunocompromised patients.
Purification and Characterization of (2R,3R)-2,3-Butanediol Dehydrogenase of the Human Pathogen Neisseria gonorrhoeae FA1090 Produced in Escherichia coli
Springer Science and Business Media LLC - Tập 63 - Trang 491-501 - 2021
Wanggang Tang, Chaoqun Lian, Yu Si, Jianrong Chang
2,3-Butanediol dehydrogenase (BDH), also known as acetoin/diacetyl reductase, is a pivotal enzyme for the formation of 2,3-butanediol (2,3-BD), a chiral compound with potential roles in the virulence of certain pathogens. Here, a NAD(H)-dependent (2R,3R)-BDH from Neisseria gonorrhoeae FA1090 (NgBDH), the causative agent of gonorrhoea, was functionally characterized. Sequence analysis indicated that it belongs to zinc-containing medium-chain dehydrogenase/reductase family. The recombinant NgBDH migrated as a single band with a size of around 45 kDa on SDS-PAGE and could be confirmed by Western blotting and mass spectrometry. For the oxidation of either (2R,3R)-2,3-BD or meso-2,3-BD, the enzyme exhibited a broad pH optimum between pH 9.5 to 11.5. For the reduction of (3R/3S)-acetoin, the pH optimum was around 6.5. The enzyme could catalyze the stereospecific oxidation of (2R,3R)-2,3-BD (Km = 0.16 mM, kcat/Km = 673 s−1 · mM−1) and meso-BD (Km = 0.72 mM, kcat/Km = 165 s−1 · mM−1). Moreover, it could also reduce (3R/3S)-acetoin with a Km of 0.14 mM and a kcat/Km of 885 s−1 · mM−1. The results presented here contribute to understand the 2,3-BD metabolism in N. gonorrhoeae and pave the way for studying the influence of 2,3-BD metabolism on the virulence of this pathogen in the future.
Advances in Pathophysiology of Triple-Negative Breast Cancer: The Potential of lncRNAs for Clinical Diagnosis, Treatment, and Prognostic Monitoring
Springer Science and Business Media LLC - Tập 63 - Trang 1093-1102 - 2021
Yangkun Zhou, Yang Yue, Siyu Fan, Qiaojun Jia, Xianfeng Ding
Recent studies have shown that long non-coding RNAs (lncRNAs) are involved in several gene expression regulation processes, including epigenetic regulation, transcriptional regulation, post-transcriptional regulation, and translation regulation. It also plays a crucial role in the regulation of several characteristics of cancer biology, and the dysregulation of lncRNA expression in cancer may be part of the cause of cancer progression. Meanwhile, more and more studies are trying to determine the association between lncRNA expression and TNBC, as well as the functional role and molecular mechanism of the abnormally expressed lncRNA. Therefore, this review lists some abnormal lncRNAs in TNBC, further analyzes their molecular mechanisms and biological roles in the development of TNBC, and summarizes the potential of lncRNAs as biomarkers and therapeutic targets of TNBC, so as to provide ideas for clinical diagnosis, targeted therapy, and prognosis monitoring of TNBC.
Molecular Cloning and Expression of a Cryptochrome Gene CiCRY-DASH1 from the Antarctic microalga Chlamydomonas sp. ICE-L
Springer Science and Business Media LLC - Tập 62 - Trang 91-103 - 2019
Xin Zhang, Zhou Zheng, Yingying He, Lina Liu, Changfeng Qu, Jinlai Miao
Cryptochromes (CRYs) are flavin-binding proteins that sense blue and near-ultraviolet light and participate in the photoreactions of organisms and the regulation of biological clocks. In this study, the complete open reading frame (ORF) of CiCRY-DASH1 (GenBank ID MK392361), encoding one kind of cryptochrome, was cloned from the Antarctic microalga Chlamydomonas sp. ICE-L. The quantitative real-time PCR study showed that the CiCRY-DASH1 had the highest expression at 5 °C and salinity of 32‰. The CiCRY-DASH1 was positively regulated by blue, yellow, or red light. Moreover, the CiCRY-DASH1 can positively respond to extreme polar day and night treatment and exhibit a certain circadian rhythm, which indicated that CiCRY-DASH1 participated in the circadian clock and its expression was regulated by circadian rhythms. And the CiCRY-DASH1 was more noticeably affected by ultraviolet-B radiation than ultraviolet-A radiation, indicating ultraviolet-B light does obvious damage to Antarctic microalgae.
Sequence-Specific Gene Silencing of acrA in the Multi-drug Efflux System AcrAB Induces Sensitivity in Drug-Resistant Klebsiella pneumoniae
Springer Science and Business Media LLC - Tập 65 Số 6 - Trang 953-960 - 2023
Priyanka Ashwath, Vijaya Kumar Deekshit, Anusha Rohit, Praveen Rai, Vankadari Aditya, Nishith Babu, Indrani Karunasagar, Akhila Dharnappa Sannejal
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