Biomedical subjects

Sheng-Li Yang

Publications and source records attributed to Sheng-Li Yang.

At least 37 records · Page 2Linked to original sources

PubMed2003

Refolding and purification of Apo2l/TRAIL produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli.

As a new member of tumor necrosis factor (TNF) superfamily, TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) was produced mainly as inclusion bodies by recombinant Escherichia coli with a temperature-inducible expression system. High concentrations of both biomass (65 g dry cells l(-1)) and inactive TRAIL (4.8 g l(-1)) were obtained by applying a high-cell-density cultivation procedure. After the inclusion bodies were washed and solubilized. TRAIL refolded when at 1 mg ml(-1) by a simple pulse dilution method with a 35% yield. Renatured TRAIL was purified to electrophoretic homogeneity by one-step immobilized metal affinity chromatography. The purified TRAIL showed strong cytotoxicity activity against human pancreatic 1990 tumor cells, with ED50 about 1.6 microg ml(-1).

Apoptosis Regulatory Proteins↗

PubMed2003

Molecular cloning, characterisation and tissue-specific expression of human LAG3, a member of the novel Lag1 protein family.

We identified and characterised the cDNA coding for human Lag3, a member of the novel eukaryotic Lag1 protein family which was related to the Saccharomyces cerevisiae longevity-assurance gene LAG1. The composite nucleotide sequence revealed a coding region of 1140 bp, a 5'-UTR of 210 bp and a 3'-UTR of 900 bp. The deduced sequence of 380 amino acids had six transmembrane regions and the conserved Lag1 motif. Northern blot analysis revealed that the approximately 2.2 kb LAG3Hs transcript was expressed in all 12 human tissues, high in kidney and liver, moderate in brain, heart, skeletal muscle, placenta and lung and low in colon, thymus, spleen, small intestine and peripheral blood leukocyte. Because of its structural similarity to LAG1, it was postulated that LAG3Hs may mediate sphingolipids metabolism.

5' Untranslated Regions↗

PubMed2003

Interaction of C17orf25 with ADP-ribose pyrophosphatase NUDT9 detected via yeast two-hybrid method.

The gene C17orf25 was isolated from the liver by RACE PCR. nudt9 gene was screened by yeast two-hybrid method in MatchMaker human HeLa cDNA library. NUDT9 is an enzyme that has pyrophosphatase activity with ADP-ribose as its substrate. Fusion expression of C17orf25 and GFP and computer analysis showed that C17orf25 was probably located in mitochondria. Furthermore, C17orf25 may suppress the cell growth by interaction with NUDT9.

Amino Acid Sequence↗

PubMed2003

Cloning and expression of adiponectin and its globular domain, and measurement of the biological activity in vivo.

3T3-L1-adipocytes produce the adipocyte complement related protein of 30 kD (ACRP30), which is exclusively expressed in differentiated adipocytes. Decreased expression of ACRP30 correlates with insulin resistance in mouse models of altered insulin sensitivity. Adiponectin, the human homologue of ACRP30, circulates in human plasma at high levels. Plasma adiponectin levels have been reported to be decreased in some insulin-resistant states, such as obesity and type II diabetes mellitus. Here, full-length adiponectin and its C-terminal globular head domain (gAdiponectin) were expressed in Escherichia coli and gAdiponectin was used to immunize a rabbit to obtain polyclonal antiserum with titer of 10,000. Adiponectin was detected in human plasma with the use of gAdiponectin anti-serum by Western blot analysis, which was also detected by gACRP30 anti-serum. Injection in alloxan-treated rats with purified recombinant fusion adiponectin or gAdiponectin transiently abolished hyperglycemia. So adiponectin and gAdiponectin might have activity as a glucose lowering agent and potentially as a therapeutic for metabolic disease. All these results suggested that the recombinant protein had biological activity, and provided a useful tool in further studies.

3T3-L1 Cells↗

PubMed2003

[The immunopotentiation of human B lymphocyte stimulator C-terminal peptide].

The cDNA of human B lymphocyte stimulator C-terminal peptide (C-BLyS) was amplified by nested PCR from cDNA library of human fetal brain. The expression plasmid pT7450-C-BLyS was constructed and transformed into E. coli BL21 CodonPlus (DE3) RIL which can recognize many rare codons. The C-BLyS protein was expressed as inclusion body in E. coli BL21 CodonPlus (DE3) RIL and the inclusion body of C-BLyS was denatured and then refolded by dialysis and purified by ion exchange chromatography. The refolded and purified C-BLyS can specifically bind with BCMA-Fc, a fusion protein of BLyS receptor and human IgG1 Fc. Furthermore, C-BLyS is proved to be an effective stimulator in mouse splenocytes proliferation in vitro and effective immunostimulant in vivo.

Animals↗

PubMed2003

[Use of PCR related methods in detection of gene mutation].

Many inherited diseases and drug resistance have been attributed to mutations in corresponding genes. In this paper, several techniques based on PCR used in diagnosis were concluded. The development and research progress of Mismatch PCR were discussed in details. Some information about an assay that we developed for detection of antimicrobial resistance to quinolones was also described.

English Abstract↗

PubMed2003

[HRCA and application in detection of genetically modified plant].

In this article primary studies of the application of hyperbranched rolling cycle amplification (HRCA) in exogenous genes detection of transgenic plants were done. Four padlock probes were designed according to the sequences of four genes/DNA fragments that are used widely in transgenic plants; part of the sequence of pKK233 was chosen as the linking part of padlock probes and a pair of HRCA primers was designed according to the sequence of linking part. Study of the specificity of ligation in HRCA with isotope labeled padlock probes indicated padlock probes could be ringed effectively only when corresponding target DNA exited in the same reaction system and could not be ringed when there was no corresponding target DNA exited. Ligation time is very different according to the characteristic of target DNA being used. 5 min to 10 min is enough if the target DNA is plasmid; 30 min to 60 min is needed if the target is genome DNA of plant because it's sequence is more complex than that of plasmid's. HRCA time was analyzed which indicated longer reaction time can obviously increase the amount of products. Quantity of enzyme in HRCA was also analyzed. Different amount of enzyme (from 0.5 unit to 4 units) can give similar result when other conditions are not changed. On the basis of the research, transgenic tobacco was detected with these four padlock probes and the results were just as prospective. In order to increase the efficiency of detection, multiplex HRCA (MHRCA)was used. In MHRCA more than one padlock probes are used at the same time in the same reaction system to detect more than one targets. Because the amplification products of MHRCA will be complex and it is almost impossible to analyze with electrophoresis, so reverse-blot is used. Detection results of transgenic tobacco with this method are the same with anticipation. Compare to MPCR method we established before MHRCA is more convenient to operate and more effective in detecting exogenous genes in transgenic plants.

Nucleic Acid Amplification Techniques↗

PubMed2003

[Study of the expression membrane protein LASS2].

A human membrane protein LASS2 (Homo sapiens longevity assurance homologue 2 of yeast LAG1), which has important physiologic functions, was expressed in three different expression systems. Only the LASS2 protein carboxyl terminal hydrophilic fragment could be expressed in the prokaryote expression system and its polyclonal antibody was produced. The full length of LASS2 protein could be expressed successfully in both eukaryotic in vitro translation system and Bacuvirus expression system: Bac-to-Bac system. SDS-PAGE analysis revealed that the molecular weight of expressed product of LASS2 was about 28 kD. The product was also proved by Western blot. This recombinant LASS2 protein was purified by metal affinity resin and the purity is above 90%.

Animals↗

PubMed2002

[Expression of histone-based fusion protein HNHG in E.coli].

Histone H1 contributes to condense nucleosome into super-structure during the transformation of chromatin into chromosome. It is shown in this rep or t that the fusion protein HNHG with the core of C-terminus of histone H1(0) expressed in BL21 (DE3) could also condense the plasmid DNA, just as histones did in nucleus. Under electron microscope, plasmid DNA condensed and supercoiled after t he addition of HNHG, in contrast to plasmid DNA control. This specific ability of the fusion protein HNHG of binding and condensing plasmids could be utilized to construct novel exogenous gene delivery systems. HNHG would be a promising candidate for gene delivery.

DNA, Superhelical↗

PubMed2002

[The PTD domain of Tat protein enhance GFP protein delivering into myeloma cell SP2/0].

In order to detect the protein delivery mediated by the PTD (protein transduction domain) of TAT Protein, a expression vector, named pT7460-GFP, was constructed by insert the PTD DNA Sequence, followed by a GFP (green fluorescent protein) gene fused in-frame, into the pT7450 vector. The TAT-GFP fusion protein was expressed in the E. coli ER2566. Most of the fusion protein was presented in the inclusion body. The protein was purified by Ni2+ affinity chromatography under denature conditions, then by a Sepharose Q column to remove urea. The soluble denatured protein was added directly to medium containing the Myeloma Cell SP2/0. It came out that the fusion protein could be detected delivered into the cells under fluorescent microscope in a short time.

Gene Products, tat↗

PubMed2001

An Expression Release System for Heterogeneous Proteins in E.coli Mediated by the kil Gene.

An expression release system for heterogeneous proteins in Escherichia coli mediated by the colicin release gene(kil gene) was constructed. The system is based on the ability of the Kil protein to release periplasmic proteins into the growth medium. beta-lactamase, an E.coli periplasmic protein, and prolyl endopeptidase (PEP), a periplasmic protein of Aeromonas punctata subsp. punctata were used as report proteins. Commonly, these two proteins are seldom released into the growth medium. The results indicated that the released amounts of the beta-lactamase and of the prolyl endopeptidase were nearly 5 fold and 4 fold than control, respectively.

Journal Article↗

PubMed2001

Construction of a Novel Engineering Strain E.coli G830,which is Adoptable to High-cell-density Fermentation.

A novel engineered strain G830 adoptable to high-cell-density fermentation by integrating bacterial hemoglobin vhb (Vitreoscilla hemoglobin gene) into thr operon in the chromosome of PA1 blocking Pta-Ack metabolic pathway through the homologous recombination between the homologous fragments of integrated plasmid and that of chromosome. The engineered strain G830 was characterized by phenotype observation, PCR, thr mutant, acetate acid detection, Western blotting and VHb activity assays. In high dentity fermentation, the cellular respiration, energy metabolism, highest bacterial density and dry bacteria weight of the G830 strain were markedly better than control strains PA1 and BL21. The expression of recombinant prolyl endopeptidase (PEP) in G830 and PA1 under the above condition was high and stable. Their growth situation and fermentation parameters were similar with their parental strains without plasmids, and resided plasmids maintained stably in those strains. It revealed that the integral VHb and acetate metabolism pathway (Pta-Ack) block improved the growth of host strain under low-dissolved-oxygen conditions, enhanced the recombinant proteins production and reduced the accumulation of acetate harmful to bacterial growth. In conclusion, the novel engineering strain G830 was adoptable to high cell density fermentation.

Journal Article↗

PubMed2001

The Thioredoxin Reductase-deficient E.coli Mutant Enhances Expression into Solution of Recombinant Proteins Containing Cys Residues.

A 3D artificial protein, a salmon calcitonin hexa-polymer, a salmon calcitonin octo-polymer and a human prourokinase, was expressed in the cytoplasma of E.coli GJ980(trxB(-)) mutant. These recombinant proteins containedcysteine residues of different length ranging from 12-22 residues. The mutation was mapped to the gene for thioredoxin reductase(trxB) and was found to eliminate the activity of this enzyme, which was thought to contribute to the sulfhydryl reducing potential of the cytoplasm. Recombinant salmon calcitonin hexapolymer, salmon calcitonin octo-polymer and human prourokinase had more soluble form in cytoplasm of GJ980 mutants than in wild-type strain, while 3D-protein, which has nocysteine residue, still remain in insoluble form. Results indicate the GJ980(trxB(-)) strain allowed the formation of disulphide bonds in the cell cytoplasm which is believed to encourage correct folding and soluble expression of the recombinant proteins.

Journal Article↗

PubMed2000

The Application of a Novel Lytic System to the Recovery of Recombinant Proteins in E.coli.

In order to efficiently recover recombinant proteins, a temperature-sensitive lytic system was constructed on the basis of the feature that T4 lysozyme disrupts the bacteria through cutting specific bond in the peptidoglycan layer of cell wall. This system was evaluated by constructing and introducing a low copy plasmid pSC-lys (pSC101 replication origin) into E.coli. The plasmid contained a temperature sensitive T4 lysozyme (LYS(ts)) gene under the control of three tandem tac promoters and the LacI repressor, which is compatible with other plasmids carrying pMB1, ColE1 replication origins, etc. Under the optimum lysis conditions, 2--5 fold condensed cultures resuspended in buffer A, beta-galactosidase, recombinant chaperone GroEL and ZZ-fusion salmon hexamic calcitonin (Cal6) in E.coli were released simply, rapidly, and quantitatively, as co-expressed with LYS(ts). The two tested recombinant proteins maintained their significant productions. Instead of other cumbersome lysising methods, this novel lytic system will be useful in recovery of recombinant proteins for further purification in the field of biotechnology.

Journal Article↗

PubMed2000

Purification of Recombinant GM-CSF/IL-3 Fusion Protein.

As a new artificial haemopoietic growth factor, GM-CSF/IL-3 fusion protein appears very promi-sing to be developed as a drug in the treatment of many diseases including cancer. The purification process of the recombinant GM-CSF/IL-3 fusion protein was studied. Similar to majority of the eukaryotic proteins, GM-CSF/IL-3 was expressed as inclusion bodies in E.coli. A series of purification steps, including cell breakage, inclusion body washing, inclusion body solubilization, protein renaturation and ion-exchange chromatography, have been set up to purify the recombinant fusion protein in an active form. Experimental results showed that the inclusion body solubility increased with increasing urea concentration. During the protein renaturation process, dialysis method was chosen to remove the denaturant urea. Stepwise decrease of urea concentration in the buffer could effectively improve the protein renaturation efficiency by reducing protein aggregation. At the same time, reduced and oxidized glutathionine were added to optimize the correct disulfide bonds formation. The recombinant protein was then purified by DEAE ion-exchange chromatography. The final protein recovery was over 30%. SDS-PAGE and reverse HPLC analysis revealed over 95% purity of the final purified recombinant protein. Therefore, a laboratory-scale purification procedure of the recombinant GM-CSF/IL-3 fusion protein was basically well established.

Journal Article↗

PubMed2000

Increase in Hydrophobicity of Signal Peptide Enhances Secretion of Penicillin G Acylase.

An artificial strong hydrophobic signal peptide (ASP), containing ten leucines in tandem in the hydrophobic core, was utilized to replace the wild type signal peptide (WTSP) of penicillin G acylase (PAC), by the fusion to its 4 pro site. PAC expression plasmids, including pKKpacdeltaSP, pKKpacWTSP, pKKpacASP, pETpacWTSP and pETpacASP, were constructed. The length and the amino- and carboxyl-terminus amino acid composition of ASP and WTSP were kept identical. The activity assay and Western-blotting analysis were used to study the effect of ASP and WTSP on the secretion of PAC in tac, T7 and dissolved-oxygen regulation expression systems, respectively. Lack of signal peptide in pKKpacdeltaSP resulted in the accumulation of 91 kD PAC precursor (without signal peptide, but with the space peptide between alpha-subunit and beta-subunit) in the cytosol, indicating that the secretion of PAC depends on the signal peptide. In BL21(pKKpacASP) cells, the PAC activity and proprecursor (with signal peptide and space peptide) processing capacity were increased by about 54% and 38.5%, respectively, in comparison with BL21(pKKpacWTSP) cells. Compared with BL21(DE3) (pETpacWTSP), however, the PAC activity and proprecursor processing capacity in BL21(DE3) (pETpacASP) were enhanced by about 69% and 43.5%, respectively. The PAC activity expressed from pETpacASP was about 67% more than that from pETpacWTSP in the dissolved-oxygen-regulated expression system GJ100. Resulting from the strong hydrophobicity of ASP, therefore, the PAC activity and proprecursor processing capacity were increased by about 63% and 41% on average, respectively, in comparison with WTSP. In conclusion, the increase in hydrophobicity of the signal peptide hydrophobic core enhanced the secretion of penicillin G acylase.

Journal Article↗

PubMed1999

Molecular Cloning and Expression of a New A Chain of beta-Bungarotoxin.

The cDNA encoding a new A chain of beta-bungarotoxin was cloned from Bungarus multicinctus. It encoded a 27-amino acid signal peptide and a mature protein of 120 amino acids. The mature protein had the same 13 cysteines as the other A chains and shared high homology with them. The cDNA encoding the PLA(2) from Naja naja atra was also cloned by the use of the same degenerate primers. The cDNAs encoding the new A chain and the PLA(2) were highly expressed in E. coli as soluble fusion proteins and purified by affinity chromatography. The two recombinant proteins achieved after digestion by X(a) factor showed weak and strong PLA(2) activity, respectively.

Journal Article↗

PubMed1999

Molecular Cloning and Expression of the cDNA for Disintegrin from Agkistrodon acutus.

In order to clone novel and valuable genes from natural resources and develop genetic engineering drugs, total RNA was extracted from the venom gland of Agkistrodon acutus by one step method. The cDNAs for low molecular weight metalloproteinase were amplified by RT-PCR and cloned into the pGEMT vector. Their sequence were determined. One of the deduced amino acid sequences included a metalloproteinase and a disintegrin (acugrin) at the carboxyl terminus. This result further confirms that metalloproteinase and disintegrin are derived from the proenzyme of snake venom metalloproeinase. Acugrin exhibits a certain amino acid homology with other disintegrins from snake venoms. Acugrin has four disulfide bonds and a consensus motif RGD. The cDNA for the acugrin was cloned into the plasmid pT7ZZa and expressed in E.coli. ZZ-acugrin fusion protein could strongly inhibit aggregation of platelet induced by ADP and the IC(50) was about 4 nmol/L.

Journal Article↗