Molecular cloning, expression, purification, and mass spectrometric characterization of 3C-like protease of SARS coronavirus

Severe acute respiratory syndrome (SARS) is an acute respiratory illness, which has broken out in China. It has been known that SARS coronavirus (SARS_CoV) is a novel human coronavirus and is responsible for SARS infection. Belonging to one of the major proteins associated with SARS_CoV, SARS 3C-like protease (SARS_3CL(pro)) functions as a cysteine protease engaging in the proteolytic cleavage of the viral precursor polyprotein to a series of functional proteins required for coronavirus replication and is considered as an appealing target for designing anti-SARS agents. To facilitate the studies regarding the functions and structures of SARS_3CL(pro), in this report the synthetic genes encoding 3CL(pro) of SARS_CoV were assembled, and the plasmid was constructed using pQE30 as vector and expressed in Escherichia coli M15 cells. The highly yielded ( approximately 15mg/L) expressed protease was purified by use of NTA-Ni(2+) affinity chromatography and FPLC system, and its sequence was determined by LC/MS with the residue coverage of 46.4%.     

Effect of synthetic oligopeptides on osteoporosis

The objective of this study was to establish the solution method of GHRPS, the synthetic oligopeptides Tyr-Gly-Gly-Phe-Met-NH2, Tyr-Gly-Gly-Phe-Met-OH, Tyr-Gly-Gly-Phe-Leu-NH2, Tyr-Gly-Gly-Phe-Leu-OH, Tyr-Gly-Gly-Phe-Gly-NH2, and Tyr-Gly-Gly-Phe-Gly-OH, to verify their effect on osteoporosis. Male ICR mice (20 +/- 2 g) were used. The intramuscular injection dose of 6.3 mg/kg prednisone induced a significant decrease of body and femur weight of the animals. The subcutaneous injection dose of 18 microg/kg synthetic peptide was not effective to prevent the decrease of body and femur weight of the animals. The subcutaneous injection dose of 6.3 mg/kg prednisone elicited a decrease in content of femur calcium and in the level of serum calcium of the animals. The subcutaneous injection dose of 18 microg/kg Tyr-Gly-Gly-Phe-Leu-NH2, or Tyr-Gly-Gly-Phe-Leu-OH, or Tyr-Gly-Gly-Phe-Gly-NH2, significantly increased the content of femur calcium and decreased the level of serum calcium of the animals. It was also observed that the subcutaneous injection dose of 18 microg/kg Tyr-Gly-Gly-Phe-Gly-OH, Tyr-Gly-Gly-Phe-Leu-OH, Tyr-Gly-Gly-Phe-Met-OH, Tyr-Gly-Gly-Phe-Met-NH2 significantly increased the content of femur phosphorous and decreased the activity of ALP of the animals.     

PCAS – a precomputed proteome annotation database resource

Background  

Many model proteomes or "complete" sets of proteins of given organisms are now publicly available. Much effort has been invested in computational annotation of those "draft" proteomes. Motif or domain based algorithms play a pivotal role in functional classification of proteins. Employing most available computational algorithms, mainly motif or domain recognition algorithms, we set up to develop an online proteome annotation system with integrated proteome annotation data to complement existing resources.     

Expression,purification and functional characterization of a recombinant scorpion venom peptide BmTXKbeta

BmTXKbeta, a scorpion toxin isolated from the Chinese scorpion Buthus martensii Karsch (BmK), was expressed as a GST fusion protein in BL21 (DE3) strain. The recombinant GST-BmTXKbeta protein was purified by affinity chromatography. When treated with enterokinase, the GST-BmTXKbeta fusion protein released an approximate 6.5kDa protein which was the expected size for correctly processed. About 2mg purified recombinant BmTXKbeta protein (rBmTXKbeta) was produced from 1l bacterial culture, using this expression and purification system. The function of rBmTXKbeta was studied on the rabbit atrial myocyte by whole-cell patch clamp technique. The results showed that rBmTXKbeta inhibited the transient outward current (I(to)) of rabbit atrial myocyte with recovery after washout and the inhibition was concentration-dependent. The rBmTXKbeta prolonged the action potential duration of rabbit atrial myocyte in a concentration-dependent manner, whereas it did not affect the action potential amplitude.     

The site-directed mutagenesis of gastrodia anti-fungal protein mannose-binding sites and its expression in Escherichia coli

Gastrodia anti-fungal protein (GAFP) displays strong inhibitory activity against certain fungal pathogens. Five GAFP analogues with different mutations at mannose-binding sites and the wild-type one were expressed and purified in Escherichia coli. The inhibitory analysis of the purified various GAFPs against the growth of Trichoderma viride indicates that single amino acid mutated-type GAFPs have inhibitory activity, but its activity is much less than the wild-type one. The double and triplicate amino acids mutated GAFPs have very low inhibitory activity. For the first time it was proved that GAFP mannose-binding sites play key role in anti-fungi process.     

A simple parameter relating sequences with folding rates of small alpha helical proteins

It is found that the helix parameter (HP), which favors clustering of non-polar residues, is linearly correlated with the logarithms of rate constants of folding of small two-state alpha-helical proteins. The definition is HP = N(H)(-1) sigma [f(i)+ (f(i-1)+f(i+1))/2], where f(i)=1 or -1, if the i'th residue is hydrophobic or hydrophilic, respectively, N(H) is the number of hydrophobic residues and the summation is taken over the hydrophobic residues.     

Secreted protein prediction system combining CJ-SPHMM,TMHMM, and PSORT

To increase the coverage of secreted protein prediction, we describe a combination strategy. Instead of using a single method, we combine Hidden Markov Model (HMM)-based methods CJ-SPHMM and TMHMM with PSORT in secreted protein prediction. CJ-SPHMM is an HMM-based signal peptide prediction method, while TMHMM is an HMM-based transmembrane (TM) protein prediction algorithm. With CJ-SPHMM and TMHMM, proteins with predicted signal peptide and without predicted TM regions are taken as putative secreted proteins. This HMM-based approach predicts secreted protein with Ac (Accuracy) at 0.82 and Cc (Correlation coefficient) at 0.75, which are similar to PSORT with Ac at 0.82 and Cc at 0.76. When we further complement the HMM-based method, i.e., CJ-SPHMM + TMHMM with PSORT in secreted protein prediction, the Ac value is increased to 0.86 and the Cc value is increased to 0.81. Taking this combination strategy to search putative secreted proteins from the International Protein Index (IPI) maintained at the European Bioinformatics Institute (EBI), we constructed a putative human secretome with 5235 proteins. The prediction system described here can also be applied to predicting secreted proteins from other vertebrate proteomes. Availability: The CJ-SPHMM and predicted secreted proteins are available at: ftp://ftp.cbi.pku.edu.cn/pub/secreted-protein/     

Multiple transporters associated with malaria parasite responses to chloroquine and quinine

Mutations and/or overexpression of various transporters are known to confer drug resistance in a variety of organisms. In the malaria parasite Plasmodium falciparum, a homologue of P-glycoprotein, PfMDR1, has been implicated in responses to chloroquine (CQ), quinine (QN) and other drugs, and a putative transporter, PfCRT, was recently demonstrated to be the key molecule in CQ resistance. However, other unknown molecules are probably involved, as different parasite clones carrying the same pfcrt and pfmdr1 alleles show a wide range of quantitative responses to CQ and QN. Such molecules may contribute to increasing incidences of QN treatment failure, the molecular basis of which is not understood. To identify additional genes involved in parasite CQ and QN responses, we assayed the in vitro susceptibilities of 97 culture-adapted cloned isolates to CQ and QN and searched for single nucleotide polymorphisms (SNPs) in DNA encoding 49 putative transporters (total 113 kb) and in 39 housekeeping genes that acted as negative controls. SNPs in 11 of the putative transporter genes, including pfcrt and pfmdr1, showed significant associations with decreased sensitivity to CQ and/or QN in P. falciparum. Significant linkage disequilibria within and between these genes were also detected, suggesting interactions among the transporter genes. This study provides specific leads for better understanding of complex drug resistances in malaria parasites.     

Tissue distribution and subcellular localization of the ClC-5 chloride channel in rat intestinal cells

ClC-5 is the Cl- channel that is mutated in Dent's disease, an X-chromosome-linked disease characterized by low molecular weight proteinuria, hypercalciuria, and kidney stones. It is predominantly expressed in endocytically active renal proximal cells. We investigated whether this Cl- channel could also be expressed in intestinal tissues that have endocytotic machinery. ClC-5 mRNA was detected in the rat duodenum, jejunum, ileum, and colon. Western blot analyses revealed the presence of the 83-kDa ClC-5 protein in these tissues. Indirect immunofluorescence studies showed that ClC-5 was mainly concentrated in the cytoplasm above the nuclei of enterocytes and colon cells. ClC-5 partially colocalized with the transcytosed polymeric immunoglobulin receptor but was not detectable together with the brush-border-anchored sucrase isomaltase. A subfractionation of vesicles obtained by differential centrifugation showed that ClC-5 is associated with the vacuolar 70-kDa H+-ATPase and the small GTPases rab4 and rab5a, two markers of early endosomes. Thus these results indicate that ClC-5 is present in the small intestine and colon of rats and suggest that it plays a role in the endocytotic pathways of intestinal cells.