Biomedical subjects

Xiaocheng Gu

Publications and source records attributed to Xiaocheng Gu.

11 recordsLinked to original sources

PubMed2006

DRTF: a database of rice transcription factors.

SUMMARY: DRTF contains 2025 putative transcription factors (TFs) in Oryza sativa L. ssp. indica and 2384 in ssp. japonica, distributed in 63 families, identified by computational prediction and manual curation. It includes detailed annotations of each TF including sequence features, functional domains, Gene Ontology assignment, chromosomal localization, EST and microarray expression information, as well as multiple sequence alignment of the DNA-binding domains for each TF family. The database can be browsed and searched with a user-friendly web interface. AVAILABILITY: DRTF is available at http://drtf.cbi.pku.edu.cn

Database Management Systems↗

PubMed2006

Protein expression, crystallization and preliminary X-ray crystallographic studies on HSCARG from Homo sapiens.

Human HSCARG has been annotated as a possible cancer related protein. Amino acid homology, although at a low percentage, suggested that HSCARG contains NmrA domain and might be a member of short chain dehydrogenase reductase superfamily. In order to investigate its structure and function, HSCARG gene has been successfully expressed and purified in E. coli. HSCARG was crystallized and diffracted to a resolution of 2.4 A on Mar225 CCD Detector at SER-CAT 22BM synchrotron source. The crystals belong to F23 space group, with unit cell parameters a=b=c=223.30A, alpha=beta=gamma=90 degrees . There are two molecules per asymmetry unit.

Amino Acid Sequence↗

PubMed2005

Soluble expression, purification, and stabilization of a pro-apoptotic human protein, CARP.

CARP is a novel pro-apoptotic protein that has been cloned and characterized in our previous report. Previous studies showed that suppression of CARP expression results in cell proliferation in several mammalian cell lines and over-expression of CARP leads to apoptosis and inhibition of proliferation in seven tumor cell lines [Liu et al., CARP is a novel caspase recruitment domain containing pro-apoptotic protein, Biochem. Biophys. Res. Commun. 293 (2002) 1396]. To obtain soluble and active form of CARP protein for further functional and structural studies, we have expressed CARP in Escherichia coli by using Gateway cloning system. Optimal induction and expression conditions were also studied. Recombinant histidine-tagged CARP was expressed in E. coli when the carp gene was subcloned into a Gateway expression vector pET21-DEST. The partially soluble recombinant CARP protein was purified to near homogeneity by a two-step FPLC procedure, first by Ni2+ affinity chromatography followed by a gel-filtration chromatography, which yielded about 10 mg protein/L culture with at least 95% purity. Two peaks were detected in the analytical gel-filtration chromatograph while only one peak corresponding to monomer of the CARP protein was left after adding 2 mM dithiothreitol (DTT). The polymers observed are likely due to the formation of intermolecular disulfide bridges. These results suggest that adding DTT is a good solution to prevent the formation of disulfide bonds and to stabilize the protein. Successfully growing crystals of the purified CARP protein also proved that we can produce well folded CARP protein in E. coli.

Apoptosis↗

PubMed2005

Systematic high-yield production of human secreted proteins in Escherichia coli.

Human secreted proteins play a very important role in signal transduction. In order to study all potential secreted proteins identified from the human genome sequence, systematic production of large amounts of biologically active secreted proteins is a prerequisite. We selected 25 novel genes as a trial case for establishing a reliable expression system to produce active human secreted proteins in Escherichia coli. Expression of proteins with or without signal peptides was examined and compared in E. coli strains. The results indicated that deletion of signal peptides, to a certain extent, can improve the expression of these proteins and their solubilities. More importantly, under expression conditions such as induction temperature, N-terminus fusion peptides need to be optimized in order to express adequate amounts of soluble proteins. These recombinant proteins were characterized as well-folded proteins. This system enables us to rapidly obtain soluble and highly purified human secreted proteins for further functional studies.

Chromosome Mapping↗

PubMed2005

Alternative IMP binding in feedback inhibition of hypoxanthine-guanine phosphoribosyltransferase from Thermoanaerobacter tengcongensis.

Crystal structures of Thermoanaerobacter tengcongensis hypoxanthine-guanine phosphoribosyltransferase (HGPRT) apoenzyme and the enzyme-inosine monophosphate (IMP) complex have been determined to 2.5A and 2.2A resolution, respectively. The active form of the enzyme was identified as a tetramer in solution and the K(i) value of IMP was measured to be 45 microM for alpha-D-phosphoribosyl-1-pyrophosphate (PRPP). Conformation of the flexible loop in T.tengcongensis HGPRT, which is involved in substrate PRPP binding, is different from that observed in phosphoribosyltransferases (PRTs). It contains a 3-10 helix, and a unique double serine repeat. This loop is ordered even in the apoenzyme and assumes a half-closed conformation. The primary magnesium ion is directly coordinated by side-chains of Glu101 and Asp102, and water molecules in the apoenzyme, suggesting a possible prerequisite role for substrate PRPP binding. Most interestingly, an alternative IMP binding mode is found in the structure of T.tengcongensis HGPRT-IMP complex. The 5'-phosphate of IMP occupies the PPi position usually seen in PRT-PRPP complexes. This new observation is consistent with the lower K(i) value of IMP and may suggest a mechanism involving multiple modes of interactions between IMP and T.tengcongensis HGPRT in product release and feedback inhibition. The structure of T.tengcongensis HGPRT is compared with those of mesophilic HPRTs, and several possible features contributing to its thermostability are elucidated. Overall, T.tengcongensis HGPRT appears to be more diverged from other PRTs.

Amino Acid Sequence↗

PubMed2005

DATF: a database of Arabidopsis transcription factors.

UNLABELLED: We have probably developed the most comprehensive database of Arabidopsis transcription factors (DATF). The DATF contains known and predicted Arabidopsis transcription factors (1827 genes in 56 families) with the unique information of 1177 cloned sequences and many other features including 3D structure templates, EST expression information, transcription factor binding sites and nuclear location signals. AVAILABILITY: DATF is freely available at http://datf.cbi.pku.edu.cn

Amino Acid Sequence↗

PubMed2005

SPD--a web-based secreted protein database.

With the improved secreted protein prediction approach and comprehensive data sources, including Swiss-Prot, TrEMBL, RefSeq, Ensembl and CBI-Gene, we have constructed secretomes of human, mouse and rat, with a total of 18 152 secreted proteins. All the entries are ranked according to the prediction confidence. They were further annotated via a proteome annotation pipeline that we developed. We also set up a secreted protein classification pipeline and classified our predicted secreted proteins into different functional categories. To make the dataset more convincing and comprehensive, nine reference datasets are also integrated, such as the secreted proteins from the Gene Ontology Annotation (GOA) system at the European Bioinformatics Institute, and the vertebrate secreted proteins from Swiss-Prot. All these entries were grouped via a TribeMCL based clustering pipeline. We have constructed a web-based secreted protein database, which has been publicly available at http://spd.cbi.pku.edu.cn. Users can browse the database via a GO assignment or chromosomal-location-based interface. Moreover, text query and sequence similarity search are also provided, and the sequence and annotation data can be downloaded freely from the SPD website.

Animals↗

PubMed2005

Applications of the double-barreled data in whole-genome shotgun sequence assembly and analysis.

Double-barreled (DB) data have been widely used for the assembly of large genomes. Based on the experience of building the whole-genome working draft of Oryza sativa L. ssp. Indica, we present here the prevailing and improved uses of DB data in the assembly procedure and report on novel applications during the following data-mining processes such as acquiring precise insert fragment information of each clone across the genome, and a new kind of low-cost whole-genome microarray. With the increasing number of organisms being sequenced, we believe that DB data will play an important role both in other assembly procedures and in future genomic studies.

Cloning, Molecular↗

PubMed2004

Structure--activity relationships of hainantoxin-IV and structure determination of active and inactive sodium channel blockers.

Hainantoxin-IV (HNTX-IV) can specifically inhibit the neuronal tetrodotoxin-sensitive sodium channels and defines a new class of depressant spider toxin. The sequence of native HNTX-IV is ECLGFGKGCNPSNDQCCKSSNLVCSRKHRWCKYEI-NH(2). In the present study, to obtain further insight into the primary and tertiary structural requirements of neuronal sodium channel blockers, we determined the solution structure of HNTX-IV as a typical inhibitor cystine knot motif and synthesized four mutants designed based on the predicted sites followed by structural elucidation of two inactive mutants. Pharmacological studies indicated that the S12A and R26A mutants had activities near that of native HNTX-IV, while K27A and R29A demonstrated activities reduced by 2 orders of magnitude. (1)H MR analysis showed the similar molecular conformations for native HNTX-IV and four synthetic mutants. Furthermore, in the determined structures of K27A and R29A, the side chains of residues 27 and 29 were located in the identical spatial position to those of native HNTX-IV. These results suggested that residues Ser(12), Arg(26), Lys(27), and Arg(29) were not responsible for stabilizing the distinct conformation of HNTX-IV, but Lys(27) and Arg(29) were critical for the bioactivities. The potency reductions produced by Ala substitutions were primarily due to the direct interaction of the essential residues Lys(27) and Arg(29) with sodium channels rather than to a conformational change. After comparison of these structures and activities with correlated toxins, we hypothesized that residues Lys(27), Arg(29), His(28), Lys(32), Phe(5), and Trp(30) clustered on one face of HNTX-IV were responsible for ligand binding.

Amino Acid Sequence↗

PubMed2003

Protein preparation, crystallization and preliminary X-ray crystallographic studies of a thermostable hypoxanthine-guanine phosphoribosyltransferase from Thermoanaerobacter tengcongensis.

Native and His-tagged mutant (L160I) hypoxanthine-guanine phosphoribosyltransferase (HGPRT) from Thermoanaerobacter tengcongensis were cloned, expressed in Escherichia coli and purified. Both proteins were crystallized with polyethylene glycol as the main precipitant at 293 K using the hanging-drop vapour-diffusion method. The crystal of native HGPRT belongs to space group C222(1), with unit-cell parameters a = 65.77, b = 137.73, c = 95.27 A, and diffracted to 2.2 A resolution on an in-house X-ray generator. The crystal of the His-tagged mutant (L160I) HGPRT belongs to the space group I222, with unit-cell parameters a = 52.21, b = 88.36, c = 93.03 A, and diffracted to 1.7 A resolution in-house.

Bacillaceae↗

PubMed2003

Cloning, purification, and characterization of thermostable hypoxanthine-guanine phosphoribosyltransferase from Thermoanaerobacter tengcongensis.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) from a newly characterized thermophile Thermoanaerobacter tengcongensis was expressed in Escherichia coli and purified. Analytical gel filtration suggested that the enzyme exist as a homotetramer in solution. The optimal pH for the forward reaction was found to be 8.0 and the optimal temperature 70 degrees C. The steady-state kinetic characteristics suggest that hypoxanthine is the most effective substrate. This enzyme showed a half-life of 75min at 50 degrees C and no apparent loss of activity after 3 months at 4 degrees C.

Amino Acid Sequence↗