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Biomedical subjects

Ju Chu

Publications and source records attributed to Ju Chu.

At least 19 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↗
PubMed2006

[The effect of induction temperature on aggregation of consensus interferon-alpha expressed by Pichia pastoris].

The effect of induction temperature on aggregation of consensus interferon-alpha expressed by Pichia pastoris was investigated. The cell growth and cIFN level were analyzed and compared when Pichia pastoris was grown at 30,25,20 degrees C during induction phase, using 5.0L fermentor. The result suggested that the cell growth was not affected much under the different induction temperature, but the protein level declined markedly with the decrease of the induction temperature. The total protein ammount induced at 20 degrees C was 67.8 percent of that at 30 degrees C. SDS-PAGE and native-PAGE as well as Western blotting analysis were further conducted. The electrophoresis results revealed that cIFN formed aggregates after secreted into media when protein was induced at 30 degrees C but this problem can be restored by decreasing the induction temperature to 20 degrees C. cIFN monomer in supernatant arrived at 570mg/L and bioactivity of fermentation broth reached 1.05 x 10(9) IU/mL at 20 degrees C of induction temperature. The amount of cIFN monomer and bioactivity in supernatant elevated 7.2 and 38.7 times, respectively, when the induction temperature was controlled at 20 degrees C instead of conventional 30 degrees C.

Fermentation↗
PubMed2005

Multi-scale methodology: a key to deciphering systems biology.

Presently, it is widely accepted complex systems couldn't be comprehended by studying parts in isolation without examining integrative and emergent properties, and system-level understanding thus has become the focus in biological science. However, it should also be noted that common systematic analysis was restricted to large-scale analysis at a certain level, while the facts that the nature of complex systems is their multi-scale structures was usually neglected or ignored. Therefore, this paper described a multi-scale methodology to investigate the nature of biological complexity and prospected this methodology could lead to a promising revolution in current system-level understanding and the integration of molecular biology databases.

Animals↗
PubMed2005

[Influence of Mn2+ on the biotechmycin fermentation].

The effect of Mn2+ on the biotechmycin fermentation by Bioengineered strain WSJ-l-195 was studied. In the fermentation process, Mn2+ could improve the biological potency significantly, especially when Mn2+ concentration was 5 mmol/L added at 24 h. The pH profile of fermentation broth decreased gradually after 5 mmol/L Mn2+ supplemented at 24 h, and PMV was lower than that of the control sample. Further research about the influence of Mn2+ on the biosynthesis of biotechmycin was carried out in the aspect of organic acids. The results showed that concentrations of organic acids in a fermentation with 5 mmol/L Mn2+ supplemented at 24 h had been changed greatly, especially the concentration of propionic acid, of which the highest value was about 6 times as that in the control sample at 84 h. In addition, it was found that the yield of biotechmycin could be improved significantly with tiny amount of propionic acid added. Therefore, it can be concluded that Mn2+ has profound influence on the biosynthesis of biotechmycin: it enriches the biotechmycin precursor pool such as propionic acid and thus improves the yield of biotechmycin.

Culture Media↗
PubMed2005

[Regulatory effects of ammonium ions on the biosynthesis of meilingmycin].

Based on the effects of different ammonium sulfate concentrations on meilingmycin biosynthesis, the results show that lower concentration of ammonium ions stimulates the biosynthesis of meilingmycin, while the concentration of higher than 5mmol/L inhibits the mycelial growth and the biosynthesis of the products. However, the sugar consumption rate increases with the elevating concentration of ammonium sulfate. On this basis, six enzymes, which are greatly related to the meilingmycin biosynthesis and the glucose metabolism, were measured and analyzed during the meilingmycin fermentation process. The results suggest that glucose-6-phosphate dehydrogenase, citrate synthase, succinate dehydrogenase and fatty acid synthase are stimulated by higher concentration of ammonium ions, while valine dehydrogenase and methylmalonyl-CoA carboxyltransferase are inhibited. From the results it follows that ammonium ions favors primary metabolism, that is, the HMP passway and the TCA circle is enhanced, as well as the source of the precursors for the biosynthesis of meilingmycin is restricted, which bring about the lower production of meilingmycin.

Ammonium Sulfate↗
PubMed2005

[Over-expression in Escherichia coli and characterization of apolipoprotein AI].

Apolipoprotein AI (apo AI), the major protein component of human high-density lipoprotein (HDL), is a single-chain polypeptide of 243 amino acids. Several epidemiological studies have shown that the plasma concentrations of HDL has the role of reverse cholesterol transport (RCT) and inversely correlated with the incidence of coronary artery disease. Because apo AI lacks post-translational modifications, it is convenient to express human apo AI in Escherichia coli expression system. However, there is a poor stability of the mRNA and the apo AI protein in E. coli, it is difficult to express mature apo AI in recombinant bacteria, moreover, even as a fusion protein, apo AI is still sensitive to degradation and can not be cleaved efficiently from the fusion tags. In contrast, proapolipoprotein AI (proapo AI, having an additional polypeptide containing the amino acids Arg-His-Phe-Trp-Gln-Gln at the amino-teminal of the mature protein) proved stable and undegraded in Escherichia coli, and therefore, in this research, an expression system of E. coli including a plasmid of P(R)P(L) tandem promoter was adapted to produce proapo AI. Furthermore, site-directed mutagenesis of the proapo AI cDNA was performed to generate a Clu8Asp mutation in the amino-terminal sequence of proapo AI which created an acid labile Asp-Pro peptide bond between amino acid 8 and 9, and permitted specific chemical cleavage to remove pro-peptide. After inducing with a shift of temperature, yields of recombinant proapo AI achieved about 40% of total cell protein and the recombinant proapo AI expressed proved as a form of inclusion body in cells, so protein need to renature. First of all, the protein was dissolved in buffer with denaturant, and renaturation was carried out on a hydrophobic interaction column (Phenyl Sepharose), ion-exchange chromatography and gel-filtration chromatography were then used to further purify the protein. The purified recombinant apo AI was detected by a set of tests including Western-blotting, Circular dichroism spectra and lipid-binding test, the results shown that recombinant apo AI has similar structural and lipid-binding properties identical to those of native plasma apo AI, which facilitates further research and application.

Apolipoprotein A-I↗
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

[Quantitation & optimization of guanosine fermentation process: prevention of NH4+ accumulation increases guanosine production by 70%].

Metabolic engineering has become a powerful tool for optimization of industrial fermentation processes. Metabolic engineering usually undergoes three steps: construction of a recombinant strain with improved properties, genetic and biochemical analysis of the strain, and identification of target for further improvement. Metabolic fluxes analysis is an important part of the biochemical analysis. Based on the law of mass conservation and assuming pseudo-steady-state for the intermediates in the metabolic pathways, we have quantitatively analyzed the time course of the flux distribution in Bacillus subtilis and used the data to reveal the nature of the so-called "40 hour" phenomenon in fermentation of guanosine, a key raw material for the synthesis of additives for human consumption and animal feeds. The phenomenon refers to the observation that guanosine production, which proceeds at high rate from 12 hour on, declines around 40 hour while consumption of glucose keeps increasing, leading to the lower yield of the nucleoside. Equations based upon the metabolic network of Bacillus subtilis consisted of EMP pathway, HMP pathway, TCA cycle, oxidative phosphorylation pathway and others reactions of the intermediates, was constructed. The equations were solved by using the quantitative data obtained in this study. The air flow and volume, concentration of oxygen and carbon dioxide in the exit-gas were monitored online; the concentration of biomass, glucose and guanosine was analyzed manually; and the concentration of acetate, citric acid, pyruvate, and 17 amino acids were HPLC quantified. The solutions of the equation were proved to be valid, as the experimental data on oxygen consumption agrees with that of predicted form the equation. The results indicated that at 40h of the fermentation process the flux of HMP pathway, which provides the precursor of the nucleoside, decreased while that of EMP pathway and the pathways that generate amino acids and organic acids increased. The shift correlated with the accumulation of NH4+ in the broth. The assimilation of NH4+ is an energy consuming process and could shift the metabolism to the energy generating EMP pathway. Accordingly, measures were taken to prevent the accumulation of NH4+. The interference indeed stopped the metabolism shift and boosted the guanosine production at 30 g/L, 70% higher than the level reported in literature.

Bacillus subtilis↗
PubMed2003

[Quantitative studies of the production phase in rHSA fermentation].

The model equations of the production phase of rHSA fermentation were derived on the base of both elemental balance and metabolic balance, then the unknown parameters of the model were estimated by multivariable optimization. The possible reasons of discrepancy of production rate between different period of fermentation were discussed. The model could preferably described the relations between different macroscopic reaction rates of the process and keys for the high-efficiency expression of HAS were deduced.

Fermentation↗
PubMed2003

[Application of element and metabolism balancing for the cultivation process with Streptomyces aureofaciens].

On the base of element and metablism balancing, the mathematical model of the cultivation process with Streptomyces aureofaciens was developed, and the unknown parameters in the model were estimated with the method of nonlinear optimization. Firstly the energetic coefficient of CTC biosynthesis was gained, which was 1.8 - 2.8 mol-ATP x C-mol(-1). The macroscopic reaction rates were predicted in the process and compared with the experimental values. The results show that the model can preferably describe the relationships between several macroscopic reaction rates in the process and can supervise the optimization of CTC fermentation process theoretically.

Chlortetracycline↗
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↗