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Biomedical subjects

Yingping Zhuang

Publications and source records attributed to Yingping Zhuang.

11 recordsLinked to original sources
PubMed2007

Optimization of transfection mediated by calcium phosphate for plasmid rAAV-LacZ (recombinant adeno-associated virus-beta-galactosidase reporter gene) production in suspension-cultured HEK-293 (human embryonic kidney 293) cells.

rAAV (recombinant adeno-associated virus) has become a very useful gene-delivery vector for gene therapy. However, it is very difficult to generate rAAV using triple transfection on a commercial scale, owing to its low productivity and inconveniently adhesive nature of its culture. An optimal suspension-culture transfection procedure was developed for rAAV-LacZ production in suspended HEK-293 cells mediated by calcium phosphate (lacZ, a reporter gene, codes for beta-galactosidase). The study showed that cytotoxicity of transfection complexes and cell aggregation in suspension culture were two key factors affecting high suspension-culture transfection efficiency. Cytotoxicity of transfection complexes was influenced effectively by mixture of Ca(2+) and plasmid DNA when their concentrations were decreased from 300 to 150 mM and from 3.0 to 1.5 microg/ml respectively, as manifested by a relatively higher cell viability after suspension-culture transfection. Moreover, the transfection efficiency was still less than 15%. In addition, we explored the disruption of cell aggregation and the control of transfection-complex size with 2.0 mM EGTA treatment for 30 min before transfection and the addition of 100 mM Mg(2+) during transfection respectively, procedures which enhanced transfection efficiency significantly, owing to more contact and endocytosis between cells and transfection complexes. Finally, the high transfection level and rAAV-LacZ titre achieved under optimized suspension-culture transfection conditions, namely 40% and 5 x 10(11) v.g. (vector genomes)/60 ml of medium respectively, is promising for the technique's application in the large-scale production of rAAV.

Adenoviridae↗
PubMed2006

The inhibition of aggregation of recombinant human consensus interferon-alpha mutant during Pichia pastoris fermentation.

Lower induction temperature and polyoxyethylene sorbitan monolaurate (Tween-20) were successfully used to inhibit the aggregation of recombinant human consensus interferon-alpha mutant (cIFN) during Pichia pastoris fermentation. When the induction temperature was decreased from 30 to 20 degrees C, the cIFN secreted into the medium was in the form of monomers instead of aggregates. The maximum specific activity at 20 degrees C was 4.04 times as high as that at 30 degrees C. There was no obvious effect on the cell growth at 20 degrees C, but the total protein level was decreased. Similar inhibition effect on cIFN aggregation was observed when 0.2 g l(-1) Tween-20 was added during induction. Furthermore, there was a synergistic effect found between induction temperature and Tween-20 on the inhibition of cIFN aggregation. The maximum specific activity with Tween-20 at 20 degrees C was 19.9-fold higher than that without Tween-20 at 30 degrees C.

Detergents↗
PubMed2006

Nucleotide mutations in purA gene and pur operon promoter discovered in guanosine- and inosine-producing Bacillus subtilis strains.

The promoter region of the pur operon, which contains 12 genes for inosine monophosphate biosynthesis from phosphoribosylpyrophosphate, and the purA gene, encoding the adenylosuccinate synthetase, were compared among wild-type and three purine-producing Bacillus subtilis strains. A single nucleotide deletion at position 55 (relative to translation start site) in purA gene was found in a high inosine-producing strain and in a high guanosine-producing strain, which correlates with the absence of adenylosuccinate synthetase activity in these strains. Within the pur operon promoter of high guanosine-producing strain, in addition to a single nucleotide deletion in PurBox1 and a single nucleotide substitution in PurBox2, there were 4 substitutions in the flanking region of the PurBoxes and 32 nucleotide mutations in the 5' untranslated region. These mutations may explain the purine accumulation in purine-producing strains and be helpful to the rational design of high-yield recombinant strains.

Adenylosuccinate Synthase↗
PubMed2006

Expression and aggregation of recombinant human consensus interferon-alpha mutant by Pichia pastoris.

A recombinant human consensus interferon-alpha mutant (cIFN) was expressed in Pichia pastoris. The maximum dry cell weight, cIFN concentration and antiviral activity were 160 g l(-1), 1.24 g l(-1) and 4.1 x 10(7) IU ml(-1), respec tively. The cIFN secreted into the medium was in the form of aggregates dominantly by non-covalent interaction and partially by disulphide bond. When the fermentation supernatant was disaggregated with 6 M guanidine hydrochloride, the antiviral activity of cIFN achieved 2.2 x 10(8) IU ml(-1).

Antiviral Agents↗
PubMed2004

A multi-scale study of industrial fermentation processes and their optimization.

In this article problems in multi-scale industrial fermentation processes are discussed. The problems are generated virtually, by using computer simulation on three different scales--the molecular scale (genetics), the cellular scale (metabolic regulation), and the reactor engineering scale. Inter-scale observation and operation are deemed to be crucial in the optimization of bioprocesses. Bioreaction engineering based on metabolic flux analysis and control is further elucidated. Optimization methodology for study of multi-scale problems in a fermentation process, based on correlation of data, and the scale-up technique for regulation of several bioprocess parameters are generalized by investigation of two typical fermentation processes. A novel bioreactor system was designed to monitor mass flux (for example substrates and (by-)products) in a fermentation process. It was successfully applied to the optimization and scale-up of an industrial fermentation process for penicillin, erythromycin, chlortetracyclin, inosine, and guanosine, and for production of recombinant human serum albumin and a malaria vaccine by use of the Pichia expression system. Substantial improvement of industrial fermentation productivity was achieved.

Bioreactors↗
PubMed2004

Ethanol evolution rate: a new parameter to determine the feeding rate for the production of avermectins by Streptomyces avermitilis.

A new parameter ethanol evolution rate (EER) was developed to aid in the determination of glucose feeding rate in avermectin production. The EER characterized the level of primary metabolism and its value was affected mainly by the supply of O2 and glucose. In an abnormal batch, over-feeding of glucose led to 2.5-fold increase of the maximum EER value compared to the normal one, and the production was thus decreased by nearly 80%. Together with other criteria, the EER helped to control utilization of substrate, so it has been successfully used to control glucose feeding in an industrial process.

Culture Media↗
PubMed2003

Fermentation process optimization of recombinant Saccharomyces cerevisiae for the production of human interferon-alpha2a.

The effects of different culture conditions on the expression level of human interferon-alpha2a (IFN-alpha2a) by using recombinant yeast were investigated in a 2.6-L jar fermentor. Appropriate supplement of glucose and the maintenance of residual glucose at a low level resulted in the reduction of ethanol formation and enhancement of the bioactivity of IFN-alpha2a to 4.9 x 106 from 3.1 x 10(6) IU/mL. When adenine was added evenly for 10-20 h of fermentation into the basal culture medium at a speed of 2 microg/mL of medium/h, OD600 was greatly increased to 24, and the protein increased to 276 mg/L. The content of ethanol generated was also reduced tremendously during the process, and as a result, 1.3 x 10(7) IU/mL of biologic activity was achieved. In the expression phase, pH had an important impact on expression level, which should be controlled at 5.5.

Adenine↗
PubMed2003

[Relationship between key enzyme activities of inosine-producing pathway and inosine accumulation].

The specific activities of key enzymes relating to the pathway of inosine synthesis of three different bacterial strains including high-yield, low-yield and wild strains were determined and compared systematically. A close relationship between inosine production and the specific activities of key enzymes was found. According to the enzyme characteristics of high-yield strain, suggestions on further strain improvement by modification of genetic engineering were proposed. Enzymology study is believed to be an effective way to make screening of high-yield strains more efficient.

Adenylosuccinate Synthase↗
PubMed2002

[Expression of recombinant human serum albumin in genetically engineered Pichia pastoris in high-density fermentation].

The optimum culture conditions of genetically engineered Pichia pastoris in shake-flask cultivation and in fed-batch fermentation were investigated respectively in this paper. It showed that the cultural period induced with 5 g/L methanol is 96 h, optimum methanol concentration is 10 g/L, and pH range is 5.72-6.59 in shake-flask culture. Although the seed inoculum amount increased, the target protein productivity per cell optical density was decreased. Their relationship fit the equeation Y = 12.941x(-0.5059) (r = 0.9789, where x is inoculating OD600, Y is protein productivity per cell optical density), we postulated that the restricting factor may be dissolved oxygen (DO) at shake-flask culture. With the 10% inoculum and 20 OD600 of seed, the lag phase of cell growth is 2.11 h in batch cultivation, and the relationship between cell optical density (Y) and culture time (t) is Y = 0.7841e0.2319t (r = 0.9936); The cell dry weight of broth reached 115-160 g/L and the maximum rHSA concentration was 3.6 g/L at the 120th at the fed-batch fermentation phase.

Fermentation↗
PubMed2002

[The optimization of guanosine fermentation based on process parameter correlation analysis].

The characteristic of Bacillus subtilis fermentation process of guanosine on 50 L fermentor was analyzed. Based on determination of on-line and off-line parameter, using correlation analysis, the technology study of physiologic regulation was combined with the metabolic flux distribution of synthesis process. The metabolic flux shift from HMP to EMP and TCA cycle during fermentation was found. The reason of the flux shift was preliminary analyzed, based on which the procedure was optimized to increase the yield of guanosine to 30 g/L.

Ammonia↗