Fu Jianlin
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Publication Expression of the dengue virus structural proteins in Pichia pastoris leads to the generation of virus-like particles(1997-08) Sugrue, R.J.; Fu, J.; Howe, J.; Chan, Y.-C.; INSTITUTE OF MOLECULAR & CELL BIOLOGY; MICROBIOLOGYWe have expressed cDNA encoding the dengue virus structural proteins in Pichia pastoris by chromosomal integration of an expression cassette containing the dengue virus structural genes (CprME). The yeast recombinant E protein migrated during SDS-PAGE as a 65 kDa protein when analysed by Western blotting and radioimmunoprecipitation, which is the expected molecular mass for correctly processed and glycosylated E protein. Treatment with endoglycosidases showed that the recombinant E protein was modified by the addition of short mannose chains. The E protein migrated with a buoyant density of 1.13 g/cm3 when analysed using sucrose density gradient centrifugation. Spherical structures with an average diameter of 30 nm, whose morphology resembles dengue virions, were observed in the purified fractions using transmission electron microscopy. Furthermore, the virus-like particles were immunogenic in animals and were able to induce neutralizing antibodies. This is the first report that expression of the structural genes of a flavivirus in yeast is able to generate particulate structures that resemble virions.Publication Full-length cDNA sequence of dengue type 1 virus (Singapore strain S275/90)(1992) Fu, J.; Tan, B.-H.; Yap, E.-H.; Chan, Y.-C.; Tan, Y.H.; DEAN'S OFFICE (MEDICINE); INSTITUTE OF MOLECULAR & CELL BIOLOGY; MICROBIOLOGYThe complete nucleotide sequence and the deduced amino acid sequence of the genome of dengue virus type 1 (Singapore strain S275/90) were determined from cDNA clones. The single-stranded, positive-sense RNA is 10,718 nucleotides in length and contains a single long open reading frame of 10,188 nucleotides encoding a polyprotein of 3396 amino acids. The genomic size and organization were found to be similar to that of other dengue virus serotypes. Both the nucleotide and deduced amino acid sequences were compared with the partial sequence of DEN1 (Nauru Island) and complete sequences of DEN2 (Jamaica), DEN3 (H87), and DEN4 (Dominica) virus genomes.Publication The mitochondrial respiratory chain controls intracellular calcium signaling and NFAT activity essential for heart formation in Xenopus laevis(2007-09) Chen, Y.; Wai, H.Y.; Fu, J.; Huang, G.; Melendez, A.J.; Ibrahim, F.B.M.; Lu, H.; Cao, X.; INSTITUTE OF MOLECULAR & CELL BIOLOGY; BIOCHEMISTRYThe mitochondrial respiratory chain (MRC) plays crucial roles in cellular energy production. However, its function in early embryonic development remains largely unknown. To address this issue, GRIM-19, a newly identified MRC complex I subunit, was knocked down in Xenopus laevis embryos. A severe deficiency in heart formation was observed, and the deficiency could be rescued by reintroducing human GRIM-19 mRNA. The mechanism involved was further investigated. We found that the activity of NFAT, a transcription factor family that contributes to early organ development, was downregulated in GRIM-19 knockdown embryos. Furthermore, the expression of a constitutively active form of mouse NFATc4 in these embryos rescued the heart developmental defects. NFAT activity is controlled by a calcium-dependent protein phosphatase, calcineurin, which suggests that calcium signaling may be disrupted by GRIM-19 knockdown. Indeed, both the calcium response and calcium-induced NFAT activity were impaired in the GRIM-19 or NDUFS3 (another complex I subunit) knockdown cell lines. We also showed that NFAT can rescue expression of Nkx2.5, which is one of the key genes for early heart development. Our data demonstrated the essential role of MRC in heart formation and revealed the signal transduction and gene expression cascade involved in this process. Copyright © 2007, American Society for Microbiology. All Rights Reserved.Publication Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein(1998-07-05) Cui, T.; Sugrue, R.J.; Xu, Q.; Lee, A.K.W.; Chan, Y.-C.; Fu, J.; INSTITUTE OF MOLECULAR & CELL BIOLOGYThe full-length dengue virus NS3 protein has been successfully expressed as a 94-kDa GST fusion protein in Escherichia coli. Treatment of the purified fusion protein with thrombin released a 68-kDa protein which is the expected molecular mass for the DEN1 NS3 protein. The identity of this protein was confirmed by Western blotting using dengue virus antisera. Two related activities of the recombinant NS3 protein were characterized, which were the binding of the protein to the 3'-noncoding region of the dengue virus RNA genome and NTPase activity. We demonstrated using a band shift assay that the DEN 1 NS3 protein could form a complex with the stem-loop structure in the 3'-noncoding region (3'-NOR), although sites outside the stem-loop may also participate in binding. Using various unlabeled homopolymeric and heteropolymeric RNAs as competitors for binding, it was further shown that the DEN1 NS3 protein exhibits preferential binding to a 94-nt RNA transcript from the 3'-NCR of the dengue virus. The NTPase activity of the recombinant DEN1 NS3 protein was characterized using a thin-layer chromatography assay. We found that the DEN1 NS3 protein possesses some aspects of NTPase activity, which are distinct from those found in other flaviviruses. Although the NS3 protein was able to utilize all four ribonucleoside triphosphates as its substrates, the NS3 protein showed a distinct preference for purine triphosphates (i.e., ATP and GTP). The addition of poly(U) did not stimulate NTPase activity in DEN1 NS3 protein, which contrasts with the reports for other flaviviral NS3 proteins. However, NTPase activity was specifically stimulated by the viral NS5 protein, which was manifested by a more than twofold increase in the rate of ATP hydrolysis and a 25% increase in the yield of ADP at the end of a 120-min reaction. These data suggest that the NTPase activity of the NS3 protein may be regulated by the vital NS5 protein during virus replication.Publication CDNA SEQUENCE OF DENGUE VIRUS SEROTYPE 1 (SINGAPORE STRAIN)(1997-12-10) TAN, YIN-HWEE; FU, JIANLIN; TAN, BOON-HUAN; YAP, EU-HIAN; CHAN, YOW-CHEONG; DEAN'S OFFICE (MEDICINE); INSTITUTE OF MOLECULAR & CELL BIOLOGY; MICROBIOLOGY-Publication CDNA SEQUENCE OF DENGUE VIRUS SEROTYPE 1 (SINGAPORE STRAIN)(1995-02-15) TAN YIN-HWEE; FU, JIANLIN; TAN, BOON-HUAN; YAP, EU-HIAN; CHAN, YOW-CHEONG; DEAN'S OFFICE (MEDICINE); INSTITUTE OF MOLECULAR & CELL BIOLOGY; MICROBIOLOGYDEN1-S275/90 (ECACC V92042111) is a new strain of Dengue virus serotype 1. The complete cDNA sequence of this virus has been cloned and protein-coding fragments thereof have been used in the construction of expression plasmids. DEN1-S275/90 in inactivated form, DEN1-S275/90 polypeptides or fusion proteins thereof can be incorporated into vaccines for immunisation against DEN1-S275/90 and other DEN1 viruses. The invention further provides diagnostic reagents e.g. labelled antibodies to DEN1-S275/90 proteins, and kits to detect DEN1 virus.Publication CDNA SEQUENCE OF DENGUE VIRUS SEROTYPE 1 (SINGAPORE STRAIN)(1993-11-11) TAN, YIN-HWEE; FU, JIANLIN; TAN, BOON-HUAN; YAP, EU-HIAN; CHAN, YOW-CHEONG; DEAN'S OFFICE (MEDICINE); INSTITUTE OF MOLECULAR & CELL BIOLOGY; MICROBIOLOGYDEN1-S275/90 (ECACC V92042111) is a new strain of Dengue virus serotype 1. The complete cDNA sequence of this virus has been cloned and protein-coding fragments thereof have been used in the construction of expression plasmids. DEN1-S275/90 in inactivated form, DEN1-S275/90 polypeptides or fusion proteins thereof can be incorporated into vaccines for immunisation against DEN1-S275/90 and other DEN1 viruses. The invention further provides diagnostic reagents e.g. labelled antibodies to DEN1-S275/90 proteins, and kits to detect DEN1 virus.Publication The production of recombinant dengue virus E protein using Escherichia coli and Pichia pastoris(1997-12) Sugrue, R.J.; Cui, T.; Xu, Q.; Fu, J.; Cheong Chan, Y.; INSTITUTE OF MOLECULAR & CELL BIOLOGYThe dengue virus envelope protein was expressed as a GST fusion protein using E. coli and P. pastoris as expression hosts. In E. coli the recombinant E protein is expressed initially as a soluble 81 kDa GST fusion protein. Treatment of the fusion protein with thrombin released a 55 kDa protein, which is the expected size for correctly processed, non-glycosylated recombinant E protein. The antiserum from animals immunised with this recombinant E protein was found to specifically recognise the dengue virus E protein in virus-infected cells, thus demonstrating the immunogenic nature of the recombinant E protein. This expression system allowed production of up to 2 mg of purified recombinant E protein from a 1 l bacterial culture. In contrast, expression of this GST fusion protein in P. pastoris is associated with extensive proteolytic degradation of the recombinant E protein. However, this proteolytic degradation was not observed in the truncated E protein sequences which were expressed. One of these recombinant fusion proteins, GST E401 was secreted into the culture medium at levels of up to 100 μg/l of growth medium.