10 results on '"Chen, Qijun"'
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2. var gene transcription and PfEMP1 expression in the rosetting and cytoadhesive Plasmodium falciparum clone FCR3S1.2
- Author
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Chen Qijun, Normark Johan, Blomqvist Karin, Moll Kirsten, Albrecht Letusa, and Wahlgren Mats
- Subjects
Arctic medicine. Tropical medicine ,RC955-962 ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background The pathogenicity of Plasmodium falciparum is in part due to the ability of the parasitized red blood cell (pRBC) to adhere to intra-vascular host cell receptors and serum-proteins. Binding of the pRBC is mediated by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a large multi-variant molecule encoded by a family of ≈60 var genes. Methods The study of var gene transcription in the parasite clone FCR3S1.2 was performed by semi-quantitative PCR and quantitative PCR (qPCR). The expression of the major PfEMP1 in FCR3S1.2 pRBC was analysed with polyclonal sera in rosette disruption assays and immunofluorecence. Results Transcripts from var1 (FCR3S1.2var1; IT4var21) and other var genes were detected by semi-quantitative PCR but results from qPCR showed that one var gene transcript dominated over the others (FCR3S1.2var2; IT4var60). Antibodies raised in rats to the recombinant NTS-DBL1α of var2 produced in E. coli completely and dose-dependently disrupted rosettes (≈95% at a dilution of 1/5). The sera reacted with the Maurer's clefts in trophozoite stages (IFA) and to the infected erythrocyte surface (FACS) indicating that FCR3S1.2var2 encodes the dominant PfEMP1 expressed in this parasite. Conclusion The major transcript in the rosetting model parasite FCR3S1.2 is FCR3S1.2var2 (IT4var60). The results suggest that this gene encodes the PfEMP1-species responsible for the rosetting phenotype of this parasite. The activity of previously raised antibodies to the NTS-DBL1α of FCR3S1.2var1 is likely due to cross-reactivity with NTS-DBL1α of the var2 encoded PfEMP1.
- Published
- 2011
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3. Prediction of solubility on recombinant expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli
- Author
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Chen Qijun, Ahuja Satpal, Ahuja Sanjay, and Wahlgren Mats
- Subjects
Arctic medicine. Tropical medicine ,RC955-962 ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background Cellular interactions elicited by Plasmodium falciparum erythrocyte membrane protein antigen 1 (PfEMP1) are brought about by multiple DBL (Duffy binding like), CIDR (cysteine-rich interdomain region) and C2 domain types. Elucidation of the functional and structural characteristics of these domains is contingent on the abundant availability of recombinant protein in a soluble form. A priori prediction of PfEMP1 domains of the 3D7 genome strain, most likely to be expressed in the soluble form in Escherichia coli was computed and proven experimentally. Methods A computational analysis correlating sequence-dependent features to likelihood for expression in soluble form was computed and predictions were validated by the colony filtration blot method for rapid identification of soluble protein expression in E. coli. Results Solubility predictions for all constituent PfEMP1 domains in the decreasing order of their probability to be expressed in a soluble form (% mean solubility) are as follows: ATS (56.7%) > CIDR1α (46.8%) > CIDR2β (42.9%) > DBL2-4γ (31.7%) > DBL2β + C2 (30.6%) > DBL1α (24.9%) > DBL2-7ε (23.1%) > DBL2-5δ (14.8%). The length of the domains does not correlate to their probability for successful expression in the soluble form. Immunoblot analysis probing for soluble protein confirmed the differential in solubility predictions. Conclusion The acidic terminal segment (ATS) and CIDR α/β domain types are suitable for recombinant expression in E. coli while all DBL subtypes (α, β, γ, δ, ε) are a poor choice for obtaining soluble protein on recombinant expression in E. coli. This study has relevance for researchers pursuing functional and structural studies on PfEMP1 domains.
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- 2006
- Full Text
- View/download PDF
4. Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli
- Author
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Chene Arnaud, Ahuja Sanjay, Flick Kirsten, Bejarano Maria, and Chen Qijun
- Subjects
Arctic medicine. Tropical medicine ,RC955-962 ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background The expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of P. falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells. Methods Several domains of PfEMP1 genes obtained from different P. falciparum strains were expressed in E. coli as GST-fusion proteins. Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage. Results and conclusions When expressed in E. coli recombinant proteins derived from P. falciparum sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies. The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation. Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality. Furthermore, these proteins were shown to be functionally active. Other factors such as temperature, pH, bacterial proteases or the codon optimization for E. coli had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct. In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons.
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- 2004
- Full Text
- View/download PDF
5. Phagocytosis-inducing antibodies to Plasmodium falciparum upon immunization with a recombinant PfEMP1 NTS-DBL1α domain
- Author
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Quintana, Maria del Pilar, primary, Angeletti, Davide, additional, Moll, Kirsten, additional, Chen, Qijun, additional, and Wahlgren, Mats, additional
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- 2016
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6. PfRON3 is an erythrocyte-binding protein and a potential blood-stage vaccine candidate antigen
- Author
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Zhao, Xin, primary, Chang, Zhiguang, additional, Tu, Zhiwei, additional, Yu, Shengchao, additional, Wei, Xiaoyan, additional, Zhou, Jianhua, additional, Lu, Huijun, additional, Jiang, Ning, additional, and Chen, Qijun, additional
- Published
- 2014
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7. var gene transcription and PfEMP1 expression in the rosetting and cytoadhesive Plasmodium falciparum clone FCR3S1.2
- Author
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Albrecht, Letusa, primary, Moll, Kirsten, additional, Blomqvist, Karin, additional, Normark, Johan, additional, Chen, Qijun, additional, and Wahlgren, Mats, additional
- Published
- 2011
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- View/download PDF
8. Prediction of solubility on recombinant expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli
- Author
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Ahuja, Sanjay, primary, Ahuja, Satpal, additional, Chen, Qijun, additional, and Wahlgren, Mats, additional
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- 2006
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9. Identification and characterization of DNA endonucleases in <italic>Plasmodium falciparum</italic> 3D7 clone.
- Author
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Jiang, Ning, Tu, Zhiwei, Zhang, Yiwei, Li, Jianping, Feng, Ying, Yang, Na, Sang, Xiaoyu, and Chen, Qijun
- Subjects
PLASMODIUM falciparum ,RISK of malaria ,ENDONUCLEASES ,DNA ,MALARIA prevention ,PREVENTIVE medicine - Abstract
Background:
Plasmodium falciparum is the most virulent parasite of the fivePlasmodium species that cause human malaria, and biological analysis of the parasite is critical for the development of novel strategies for disease control. DNA endonucleases are important for maintaining the biological activity, gene stability of the parasite and interaction with host immune systems. In this study, ten sequences of DNA endonucleases were found in the genome ofP. falciparum 3D7 clone, seven of them were predicted to contain an endonuclease/exonuclease/phosphatase (IPR005135) domain which plays an important role in DNA catalytic activity. The seven DNA endonucleases ofP. falciparum were systematically investigated. Methods:Plasmodium falciparum 3D7 clone was cultured in human O+ RBCs, RNA was extracted at 8, 16, 24, 32, 40, and 48 h post invasion and real-time quantitative PCR was carried out to analyse the transcription of the seven DNA endonuclease genes in asexual stages. Immunofluorescence assay was carried out to confirm the location of the encoded proteins expressed in the erythrocytic stages. Finally, the catalytic activity of the DNA nucleases were tested. Results: Of the seven proteins analysed, two proteins were not soluble. Fragments derived from the rest five endonuclease sequences were successfully expressed as soluble proteins, and which were used to generate antisera for protein localization. The proteins were all located in the nucleus at ring and trophozoite stages. While at schizont stage, proteins encoded by PF3D7_1238600, PF3D7_0107200 and PF3D7_0319200 were in the punctuated forms in the parasite most likely around nuclei of the merozoites. But the proteins encoded by PF3D7_0305600 and PF3D7_1363500 were distributed around the infected erythrocyte membrane. The enzymatic activity of the recombinant GST-PF3D7_1238600 was very efficient without divalent iron, while the activity of the rest four enzymes was iron dependent. Further, divalent irons did not show any specific enhancement on the activity of GST-PF3D7_1238600, but the activity of GST-PF3D7_0107200, GST-PF3D7_1363500 and GST-PF3D7_0319200 were Cu2+ dependent. The activity of GST-PF3D7_0305600 was dependent on Mg2+ and Mn2+ . Except GST-PF3D7_1363500, four of the GST tagged recombinant proteins hydrolysed the supercoiled circular plasmid DNA with or without divalent metal ions. The GST-PF3D7_1363500 protein only changed the supercoiled circular plasmid DNA into nicked plasmids, even with Cu2+ . Conclusions: Fragments derived from five of the endonuclease sequences ofP. falciparum 3D7 clone were successfully expressed. The proteins displayed diverse cell distribution, biochemical and enzymatic activities, which indicated that they carried different biological function in the development of the parasite in the erythrocytes. The DNA repair and DNA degradation capacity of the DNA endonucleases in the biology of the parasite remained further studied. [ABSTRACT FROM AUTHOR]- Published
- 2018
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10. Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli.
- Author
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Flick K, Ahuja S, Chene A, Bejarano MT, and Chen Q
- Subjects
- Animals, Antigens, Protozoan genetics, Antigens, Protozoan metabolism, Blotting, Western methods, Codon genetics, Escherichia coli genetics, Escherichia coli growth & development, Genome, Protozoan genetics, Heparin metabolism, Plasmodium falciparum chemistry, Protozoan Proteins metabolism, Receptors, Cell Surface genetics, Receptors, Cell Surface metabolism, Recombinant Proteins biosynthesis, Recombinant Proteins genetics, Gene Expression genetics, Molecular Biology methods, Plasmodium falciparum genetics, Protozoan Proteins biosynthesis, Protozoan Proteins genetics
- Abstract
Background: The expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of P. falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells., Methods: Several domains of PfEMP1 genes obtained from different P. falciparum strains were expressed in E. coli as GST-fusion proteins. Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage., Results and Conclusions: When expressed in E. coli recombinant proteins derived from P. falciparum sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies. The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation. Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality. Furthermore, these proteins were shown to be functionally active. Other factors such as temperature, pH, bacterial proteases or the codon optimization for E. coli had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct. In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons.
- Published
- 2004
- Full Text
- View/download PDF
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