Biomedical subjects

Sheng Yang

Publications and source records attributed to Sheng Yang.

42 records · Page 3Linked to original sources

PubMed2003

[Expression and purification of recombinant SARS coronavirus spike protein].

A novel coronavirus (SARS-coronavirus, SARS-CoV) was discovered in association with cases of severe acute respiratory syndrome (SARS) recently. The first step in coronavirus infection is binding of the viral spike protein to certain receptor on host cells. The spike protein is the main surface antigen of the coronavirus and there should be antibodies against spike protein in patients serum. Thus, to develop and expression protein fragment from spike protein gene are the purposes of this experiment. Partial spike gene fragments (751-1925 bp, 2005-3410 bp, 1-1925 bp and 32-3659 bp) and its intact gene were cloned into pET32 or pGEX vectors, and transformed into competent Escherichia coli BL21(DE3) (pLysS), respectively. 63, 78, 98, 160 and 164 kD fusion proteins were successfully expressed with amounts of 35%, 34%, 24%, 17% and 5% of total cell protein. The soluble parts of the cell crude extract were then partially purified by GST affinity chromatography.

Blotting, Western↗

PubMed2003

[Expression, purification and identification of recombinant SARS coronavirus membrane protein].

A novel coronavirus (SARS-coronavirus, SARS-CoV) was discovered as the pathogen of the severe acute respiratory syndrome (SARS). According to studies with other coronaviruses, the membrane protein (M protein) is the main structural protein and the recombinant M protein may be useful as an antigen for detecting antibodies against coronavirus and for preparing vaccine. In this work, the M protein of SARS-CoV was expressed in E. coli as fusion protein with maltose binding protein at N-terminus and MxeGyrA intein CBD at C-terminus. The recombinant protein was identified by Western blot and mass spectrometry. The soluble parts of the cell crude extract were then partially purified by MBP affinity chromatography. The purified protein will be used for the studies on M protein's structure and the development of diagnostic method of SARS.

Amino Acid Sequence↗

PubMed2002

Free radicals, antioxidants, and nutrition.

Radiation hazards in outer space present an enormous challenge for the biological safety of astronauts. A deleterious effect of radiation is the production of reactive oxygen species, which result in damage to biomolecules (e.g., lipid, protein, amino acids, and DNA). Understanding free radical biology is necessary for designing an optimal nutritional countermeasure against space radiation-induced cytotoxicity. Free radicals (e.g., superoxide, nitric oxide, and hydroxyl radicals) and other reactive species (e.g., hydrogen peroxide, peroxynitrite, and hypochlorous acid) are produced in the body, primarily as a result of aerobic metabolism. Antioxidants (e.g., glutathione, arginine, citrulline, taurine, creatine, selenium, zinc, vitamin E, vitamin C, vitamin A, and tea polyphenols) and antioxidant enzymes (e.g., superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidases) exert synergistic actions in scavenging free radicals. There has been growing evidence over the past three decades showing that malnutrition (e.g., dietary deficiencies of protein, selenium, and zinc) or excess of certain nutrients (e.g., iron and vitamin C) gives rise to the oxidation of biomolecules and cell injury. A large body of the literature supports the notion that dietary antioxidants are useful radioprotectors and play an important role in preventing many human diseases (e.g., cancer, atherosclerosis, stroke, rheumatoid arthritis, neurodegeneration, and diabetes). The knowledge of enzymatic and non-enzymatic oxidative defense mechanisms will serve as a guiding principle for establishing the most effective nutrition support to ensure the biological safety of manned space missions.

Antioxidants↗

PubMed2002

[The pH-dependent catalytic reaction of penicillin G acylase and its mutants].

The pH-dependence in the catalytic reaction of recombinant penicillin G acylase and its mutants from B.megaterium has been studied by using kinetic methods. pK(1) and pK(2)of the residues of the wild type penicillin G a cylase, involved in the catalyzed reaction, were 5.50-5.87 and 10.73, respectively, from the curves of logV(m) and log(V(m)/K(m)) versus pH. Results showed tha t the pK(1) and pK(2) values of these residues of the mutants were similar to that of the wild type. pK(1) of 5.64-5.86 for mutant A and 5.69-6.96 for mutant B were obtained, while pK(2) was 10.61 and 10.48 for mutant A and B, respectively. At the same time, pK values at different temperatures were investigated. The ionization enthalpies(deltaH) were 44.38-59.03 kJ/mol and 147.37 kJ/mol, respectively, from th e curve of pK versus temperature. It was presumed according to the results mentioned above that the ionizing residues, involved in the reaction, wer e histidine and lysine that are localized around the active site.

Bacillus megaterium↗

PubMed2000

Enhancing Penicillin G Acylase Stability by Site-directed Mutagenesis.

To improve the stability of penicillin G acylase(PGA) from Bacillus megaterium, a three-dimensional model of B.megaterium PGA was constructed based on crystal structure of penicillin G acylase from E.coli using PMODELING program. The mutation of Lys at beta427 and 430 to Ala was predicted to enhance the stability of PGA in acidic or organic solvent environment. The results showed that 2 mutant PGA had similar specific activity and Km as the parent PGA. Their optimum pH dropped 0.5 pH units. The stability of Lys430Ala was enhanced obviously at pH 5.2. The half lives of Lys427Ala and Lys430Ala were improved by 60 % and 166 %, respectively, in comparison with the parent PGA.

Journal Article↗

PubMed1999

High Expression of Penicillin G Acylase Gene from Bacillus megaterium in Bacillus subtilis.

The penicillin G acylase gene (pga) amplified by PCR from Bacillus megaterium was subcloned into an expressing vector pPZW103 (P43 as promoter). The recombinant plasmid was transferred into Bacillus subtilis DB104. Penicillin G acylase production in the B. subtilis transformant was 3-6 u/ml, higher than that of published recombinant strains. Penicillin G acylase production was induced by phenylacetic acid in B. megaterium, whereas the enzyme was produced constitutively in the B. subtilis transformant carrying B. megaterium pga. The recombinant strain showed high stability in antibiotic-free medium for 10 days. Enzyme in crude broth was purified by Al(2)O(3) chromatography and phenyl-Sepharose CL-4B hydrophobic chromatography and the total yield is 79%. The purified enzyme with specific activity of 52 u/mg can be directly immobilized for use.

Journal Article↗