[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. Subsequently, positive clone was selected by G418 and PCR, and its phenotype was determined by SDS-PAGE and MTT assay. The results demonstrated that EGFRi-IL-24 protein was expressed and shown to have the potential for use in researches of its biological function and in clinical application.

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 3.8-fold, with a specific activity of 16530 U mg(-1). As analyzed by SDS-PAGE, the molecular mass of the expressed protease was about 35 kDa, and the optimal temperature and pH of the protease were 65 degrees C and 7.5, respectively. Moreover, it still had about 80% activity after 1 h reaction at 65 degrees C.

[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. Deletion analysis of the sequence demonstrated that 414 bp to 619 bp upstream of the TSS showed predominant promoter activity, and a 105 bp length sequence can serve as this function. Additionally, a representative Sec-type signal peptide structure was detected in PB92 AprE signal peptide. By cloning the PaprE and alkaline protease signal peptide gene into pBE2, an expression vector pBEAC was constructed, and a plant sweet protein monellin gene was highly expressed in B. subtilis 1A751.

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. The hydrocortisone conversion rate of C. lunata KA-91 was increased by 42.1% compared to the original strain CL-114 at the substrate 17alpha-hydroxypregn-4-en-3, 20-dione-21-acetate addition concentration of 1.0 g/L. The by-products produced by KA-91 were fewer than those of the original strain. It was assumed that the higher cytochrome P450 content of ketoconazole-resistance mutant resulted in the increase of 11beta-hydroxylation capacity. The culture conditions for biotransformation of 17alpha-hydroxypregn-4-en-3, 20-dione-21-acetate to hydrocortisone were optimized by response surface methodology. Plackett-Burman design was applied to elucidate the key factors affecting the hydrocortisone production, and the results indicated that glucose, initial pH, and glucose to total nitrogen sources ratio (omega) had significant effects on hydrocortisone production. Box-Behnken design was employed to search for the optimal parameters of those three key factors. According to the model, the trial checking at the optimal conditions showed a high hydrocortisone conversion rate of 82.67%.

[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. The GenBank accession number was DQ202401. Lumbrokinase F238 comprised 738bp and included an open reading frame that encoded a polypeptide of 245 amino acid residues, containing a signal peptide of 7 amino acid residues and a mature peptide of 238 amino acid residues. Both nucleotide and amino acid sequences homologies were 99% after the sequence was compared with Lumbricus rubellus F-III-2. There were two base pair mutations, which subsequently caused two amino acid mutations. The characteristics and structure of F238 was analysed and predicted with biology softwares and databases. The pl of F238 was 4.61. It had eleven Cysteines, which formed three disulfide bonds. Its secondary structure mainly consisted of beta-sheet. Lumbrokinase F238 had serine active center. It was a protease in trypsin family of serine protease superfamily. Lumbrokinase gene F238-m without signal peptide sequence was obtained by PCR using pUCm-T-F238 as template. The expression vector pPIC9-F238-m was constructed by inserting gene F238-m into yeast expression and secretion plasmid pPIC9. Plasmid pPIC9-F238-m was linearized with BgIII and then transformed into Pichia pastoris strain GS115 cell by electroporation method. Phenotypes of transformants were screened in MM and MD plates to ensure the integration of lumbrokinase gene F238-m into yeast chromosome DNA. Methanol was added to a final concentration of 0.5% for the expression of recombination protein every 24h to maintain induction. The result of SDS-PAGE showed that the molecular weight of the expression product was about 28 kDa, in correspondence with the theoretical molecular weight. After the induction of expression, the fibrinolytic activity of the supernatant was measured using artificial fibrin plates. Then the engineering strain of high activity was obtained, and the fibrinolytic activity was up to 100 U/mL.

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. Glucose and pH were identified as having significant effects (with confidence level >90%). Glucose concentration and initial pH were subsequently optimized by use of a central composite design. The result indicated that glucose and pH had a significant interactive effect on natamycin production. The optimal glucose concentration and initial pH value were 38.2 g/L and 7.8, respectively. This optimization strategy led to a natamycin yield of 2.45 g/L, which was nearly 90% higher than that in the original medium.

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, MgSO4.7H2O and CaCl2.2H2O are important for avilamycin production. A polynomial model related to medium components and avilamycin yield had been established. A high coefficient of determination (R2 = 0.92) was obtained that indicated good agreement between the experimental and predicted values of avilamycin yield. Student's T-test of each coefficient showed that all the linear and quadratic terms had significant effect (P > |T| < 0.05) on avilamycin yield. The significance of tested components was related to MgSO4.7H2O (0.37 g/L), CaCl2.2H2O (0.39 g/L), soybean flour (21.97 g/L) and soluble starch (37.22 g/L). The yield of avilamycin reached 88.33 +/- 0.94 mg/L (p < 0.05) that was 2.8-fold the initial yield.

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. When the concentrations of glucose, ammonium ions, and phosphate in the basal medium exceeded 20 g/L, 10 mmol/L, and 10 mmol/L, respectively, avilamycin biosynthesis greatly decreased. When 20 g/L glucose was added at 32 h, avilamycin yield decreased by 70.2%. Avilamycin biosynthesis hardly continued when 2-deoxy-glucose was added into the basal medium at 32 h. There was little influence on avilamycin biosynthesis with the addition of the 3-methyl-glucose (20 g/L) at 32 h. In the presence of excess (NH4)2SO4 (20 mmol/L), the activities of valine dehydrogenase and glucose-6-phosphate dehydrogenase were depressed 47.7 and 58.3%, respectively, of that of the control at 48 h. The activity of succinate dehydrogenase increased 49.5% compared to the control at 48 h. The intracellular adenosine triphosphate level and 6-phosphate glucose content of S. viridochromogenes were 128 and 129%, respectively, of that of the control at 48 h, with the addition of the 40 mmol/L of KH2PO4. As a result, high concentrations of glucose, ammonium ions, and inorganic phosphate all led to the absence of the precursors for avilamycin biosynthesis and affected antibiotic synthesis.

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. The extracellular laccase activity of the PMAD11 recombinants was found to be 1.3-fold higher than that of the PMAD16 recombinants. The identity of the recombinant protein was further confirmed by immunodetection using the Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0 kDa, similar to that of the native form. The effects of copper concentration, cultivation temperature, pH and methanol concentration in the BMMY on laccase expression were investigated. The laccase activity in the PMAD11 recombinant was up to 12.6 U ml(-1) by optimization.

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. coli so as to optimize its expression. Monellin was produced and accounted for 45% of total soluble proteins. It was purified to yield 43 mg protein per g dry cell wt. The purity of the recombinant protein was confirmed by SDS-PAGE.

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. subtilis cells. The extracellular alpha-amylase activities in two recombinants were 1001 and 2012 U ml(-1), respectively. The purity of the recombinant product was confirmed by SDS-PAGE.

[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. The enzyme hydrolyzed N-Succinyl-Ala-Ala- Pro-Phe-pNA, and Km was 0.23 mmol/L and Kcat 16.36 s(-1). The optimal temperature of the enzyme for hydrolying fibrin was 45 degrees C, and the optimal pH range of 6.8 - 8.8. The isoelectric point of the enzyme estimated by isoelectric focusing electrophoresis was 8.5 +/- 0.1. The enzyme was a glycoprotein. EDTA, PCMB, PMSF inhibited the activety of the enzyme, and SBTI, Lys, TPCK, Aprotinine had none obvious inhibition, which suggested that the activity centre of the enzyme had hydrosulfuryl, metal and serine. The first 12 amino acids of the N-termimal sequence of the enzyme were NH2-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly, and had none homology with that of other fibrinolytic enzyme from other microbes. The novel fibrinolytic enzyme from Rhizopus chinensis 12# has potential to become a therapeutic agent in thrombosis.

[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. Under the expressing conditions, monellin was produced accounting for 44.8% of total soluble proteins. The E. coli-expressed single-chain monellin is 3000 times sweeter than sucrose. The thermal-stability and acid-resistance of the protein are higher than the natural monellin.

[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. The effect of CPS on the cell immunoactivity was studied in detail, the results show that CPS possesses bidirectional immunoregulation on the spleen cells of mice, that is, low concentration of CPS can stimulate the immune response while the high concentration manifests the inhibition significantly. The investigation results will benefit on the exploitation of the CPS.

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. The laccase activity obtained with the Saccharomyces cerevisiae alpha-factor signal peptide was found to be twofold higher than that obtained with the native secretion signal peptide. The extracellular laccase activity in recombinants with the alpha-factor signal peptide was 9.79 U ml(-1). The presence of 0.2 mM copper was necessary for optimal activity of laccase. The expression level was favoured by lower cultivation temperature. The identity of the recombinant protein was further confirmed by immunodetection using Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0 kDa, similar to that of the native form.

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). The purity of the recombinant product was confirmed by SDS-PAGE.