[Construction of eukaryotic expression vector of EGFRi-IL-24 recombinant gene].

The epithelial growth factor receptor interference (EGFRi) was obtained by synthetic primers. Overlapping PCR was used to produce EGFRi-IL-24 fusion gene, which is linked by Gly4Ser3. After sequence analysis, EGFRi-IL-24 was cloned into expression vector pPIC9k; EGFRi-IL-24/pPIC9k was linearized with SacI,and then transformed to electroporated pastoris GS115.

[Cloning, characterization and application of the promoter region of the alkaline protease gene in Bacillus alcalophillus PB92].

Promoter function fragment of alkaline protease gene (GenBank accession number: EU130686) was cloned from Bacillus alcalophillus PB92 genome by TAIL-PCR. Sequenced and analyzed revealed that it contains several typical promoter characterized regions. Two reverse translation frames were located in -538~-370 bp and-275~-128 bp.

Natamycin production by Streptomyces gilvosporeus based on statistical optimization.

Natamycin has been widely used as a natural preservative to prevent mold contamination in food. In this study, statistically based experimental designs were employed for the optimization of medium components for natamycin production by Streptomyces gilvosporeus. After glucose, yeast extract, and soy peptone were screened as suitable carbon and nitrogen sources, a full factorial design was used to evaluate the effects of various factors on natamycin production.

Expression, purification, and characterization of a thermophilic neutral protease from Bacillus stearothermophilus in Bacillus subtilis.

The gene coding for a thermophilic neutral protease from Bacillus stearothermophilus was expressed in Bacillus subtilis DB104, under the control of the sacB gene promoter. This was followed by either the native signal peptide sequence of this protease or the signal peptide sequence of the sacB gene. The protease was purified 3.

Optimisation of hydrocortisone production by Curvularia lunata.

A new method for breeding the hydrocortisone overproducing strain Curvularia lunata by screening ketoconazole-resistance mutant was developed. A hydrocortisone overproducing mutant C. lunata KA-91 with ketoconazole-resistance marker was obtained from protoplasts treated with ultraviolet radiation.

Statistical optimization of medium components for avilamycin production by Streptomyces viridochromogenes Tü57-1 using response surface methodology.

A fermentation medium for avilamycin production by Streptomyces viridochromogenes Tü57-1 has been optimized. Important components and their concentrations were investigated using fractional factorial design and Box-Behnken Design. The results showed that soybean flour, soluble starch, MgSO4.

[Cloning and expression of lumbrokinase gene in Pichia pastoris].

Lumbrokinase gene F238 was amplified by RT-PCR from the total RNA of earthworm (Eisenia fetida). The gene including signal peptide sequence was inserted into pUCm-T vector to construct pUCm-T-F238. The product was sequenced.

Regulation of avilamycin biosynthesis in Streptomyces viridochromogenes: effects of glucose, ammonium ion, and inorganic phosphate.

Effects of glucose, ammonium ions and phosphate on avilamycin biosynthesis in Streptomyces viridochromogenes AS4.126 were investigated. Twenty grams per liter of glucose, 10 mmol/L ammonium ions, and 10 mmol/L phosphate in the basal medium stimulated avilamycin biosynthesis.

High-level expression of a synthetic gene encoding a sweet protein, monellin, in Escherichia coli.

The expression of a synthetic gene encoding monellin, a sweet protein, in E. coli under the control of T7 promoter from phage is described. The single-chain monellin gene was designed based on the biased codons of E.

Optimization of the expression of a laccase gene from Trametes versicolor in Pichia methanolica.

A cDNA encoding for laccase (Lcc1) was isolated from the ligninolytic fungus Trametes versicolor by reverse transcriptase polymerase chain reaction. The Lcc1 gene was subcloned into the Pichia methanolica expression vector pMETalphaA and transformed into the P. methanolica strains PMAD11 and PMAD16.

Expression and secretion of an acid-stable alpha-amylase gene in Bacillus Subtilis by SacB promoter and signal peptide.

Alpha amylase gene from Bacillus licheniformis was mutated by site-directed mutagenesis to improve its acid stability. The mutant gene was expression in Bacillus subtilis under the control of the promoter of sacB gene which was followed by either the alpha-amylase leader peptide of Bacillus licheniformis or the signal peptide sequence of sacB gene of Bacillus subtilis. Both peptides efficiently directed the secretion of alpha-amylase from the recombinant B.

[Overexpression of a sweet protein monellin in Escherichia coli].

According to the amino acid sequence of monellin, a single chain 294bp monellin gene was synthesized and inserted into vector pET-22b to yield the recombinant secretion plasmid pETMO. The single-chain monellin gene was designed based on the biased codons of E. coli so that its expression would be then optimized.

[Extraction and purification of the Klebsiella pneumoniae capsular polysaccharide and the effection on the cell immunoactivity].

Klebsiella pneumoniae was cultured followed by the preparation and immunoactivity elucidating of its polysaccharide (CPS). The lysis of cell is the first key step in the preparation, under the co-action of trypsin, lysozyme and NP-40, the cell lysed within 2h, then the lysate was concentrated by ultrafiltration which serves as concentrating and partial purifying action simultaneously. Crude CPS was got by ethanol precipitation, then purified through the Ion-exchange and gel filtration, the purity of CPS was judged by the gel filtration and agarose gel electrophoresis.

[Characteristics of a new fibrinolytic enzyme produced from Rhizopus chinensis 12#].

As a therapeutic agent in thrombosis the fibrinolytic enzymes are of interest and the search for a new enzyme continues. A novel fibrinolytic enzyme was produced from Rhizopus chinensis 120, which was screened from the starter for brewing rice wine in the South of China, by solid fermentation, and purified through ammonium sulfate precipitation, hydrophobic interaction, ionic exchange and gel filtration chromatographies. The purified enzyme hydrolyzed fibrin, it cleaved the alpha-, beta- and gamma-chains of fibrinogen simultaneously, and it also activated plasminogen to plasmin.

Molecular cloning of the cDNA encoding laccase from Trametes versicolor and heterologous expression in Pichia methanolica.

A cDNA encoding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene Lcc1, which encodes a laccase isoenzyme of 498 amino acid residues preceded by a 22-residue signal peptide. The Lcc1 cDNA was cloned into the vectors pMETA and pMETalphaA and expressed in Pichia methanolica.

Purification and characterization of a novel fibrinolytic enzyme from Rhizopus chinensis 12.

A novel fibrinolytic enzyme from Rhizopus chinensis 12 was purified through ammonium sulfate precipitation, hydrophobic interaction, ionic exchange, and gel filtration chromatography. The purification protocol resulted in a 893-fold purification of the enzyme, with a final yield of 42.6%.

Heterologous expression of lignin peroxidase of Phanerochaete chrysosporium in Pichia methanolica.

The cDNA encoding for lignin peroxidase of Phanerochaete chrysosporium was expressed in the Pichia methanolica under the control of the alcohol oxidase (AUG1) promoter which was followed by either the lignin peroxidase leader peptide of Phanerochaete chrysosporium or the Saccharomyces cerevisiae alpha-factor signal peptide. Both peptides efficiently directed the secretion of lignin peroxidase from the recombinant yeast cell. The extracellular lignin peroxidase activity in two recombinants was 932 U l(-1) and 1933 U l(-1).