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Effects of different methanol feeding strategy on hirudin production in high-density fermentation by recombinant Pichia pastoris.

Xiang-Shan ZHOU ; Wei-Min FAN ; Yuan-Xing ZHANG

Chinese Journal of Biotechnology.2002;18(3):348-351.

Four different methanol feeding modes were evaluated in the hirudin production in high-density fermentation by Pichia pastoris. It was difficult to avoid methanol excessive in the broth with the feeding strategy only based on DO level. On the other hand, the fluctuation in methanol concentration was observed with methanol feeding strategy by off-line gas chromatography. However, the stable methanol concentration was perfectly achieved by the on-line monitoring with methanol sensor. The supply of energy was improved by feeding glycerol at a limited rate as well as methanol in the induction phase. Therefore, the high cell dry weight (162 g/L) and high hirudin activity (2.4 x 10(4) ATU/mL or 1.7 g/L) was obtained in the fed-batch fermentation of recombinant Pichia pastoris by methanol-glycerol mixed feeding.
Fermentation ; Glycerol ; pharmacology ; Hirudins ; biosynthesis ; Methanol ; pharmacology ; Pichia ; genetics ; metabolism ; Recombination, Genetic

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In vitro culture of umbilical cord blood MNC and CD34+ selected cells.

Bin WANG ; Zi-Zhen KANG ; Zhan-You CHI ; Wen-Song TAN

Chinese Journal of Biotechnology.2002;18(3):343-347.

For in vitro studies, both CD34+ selected cell and mononuclear cell (MNC) can be used to expand hematopoietic stem/progenitor cells. To investigate the expansion characteristics of mononuclear cells (MNC) and CD34+ selected cells the two cell fractions were cultured in the medium containing cytokine cocktails of SCF + IL-3 + IL-6 + FL + Tpo. It was found that the CD34+ selected cells had presented a high proliferation potential. The expansion of CD34+ selected cells could be maintained for 8 weeks while that of MNCs declined after 4 weeks. During the culture period, the maximum expansion of total cells in CD34+ selected cell culture achieved 31,270.9 +/- 8640.5 times, while that of MNC reached 50.9 +/- 8.2 times only. In the culture of MNCs, the colony density and the proportion of CD34+ cells increased from day 0 to day 7. However, in the culture of CD34+ selected cells, both the colony density and the proportion of CD34+ cells declined continuously during the whole culture period. During the ex vivo culture of CD34+ selected cells, the maximum expansion of CFU-GM and CD34+ cells achieved 185.7 +/- 14.1 fold and 191.7 +/- 188.8 fold, respectively. They are much higher than that of MNC, which were 12.4 +/- 3.2 fold and 50.6 +/- 33.2 fold only. While the BFU-E of both cell fractions only expanded by few times, which were 7.2 +/- 5.2 and 10.1 +/- 3.4 times, respectively. The results showed that the CD34+ selected cells culture could obtain more CFU-GM cells and CD34+ cells during the whole culture period.
Antigens, CD34 ; analysis ; Cell Count ; Cell Separation ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; physiology ; Leukocytes, Mononuclear ; cytology

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Site-directed mutation of PoIFN-alpha and its expression in Escherichia coli.

Tao CHEN ; Rui-Song YU ; Hui-Li LIU ; Zhen LI ; Xiang-Rong CAO

Chinese Journal of Biotechnology.2002;18(3):339-342.

By using huge primer PCR Cys86 (TGC) of PoIFN-alpha was mutated to Tyr(TAC), and the first code TGT was simultaneously changed to TGC, which is a bias code of E. coli. The expression plasmid pGEX-IFN was constructed successfully. Recombinant porcine IFN alpha, which is expressed as inclusion bodies, was about 20% of the total proteins. The inclusion body was dissolved in 8 mol/L urea and subsequently renatured by dilution in refolding buffer. In order to obtain pure protein, the renatured IFN alpha was purified by FPLC, and the cytokine activity (5200 IU/mg) was verified by inhibiting the cytopathic effect.
Escherichia coli ; genetics ; Interferon-alpha ; biosynthesis ; isolation & purification ; Mutagenesis, Site-Directed ; Protein Precursors ; biosynthesis ; isolation & purification ; Recombinant Proteins ; biosynthesis ; isolation & purification

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A novel resolution vector with Bacillus thuringiensis plasmid replicon ori44.

Lan WU ; Ming SUN ; Chen-Guang ZHU ; Lei ZHANG ; Zi-Niu YU

Chinese Journal of Biotechnology.2002;18(3):335-338.

The resolution recognization sites of transposon Tn4430 of Bacillus thuringiensis was inserted into cloning vector pRSET B and pUC19, resulting recombinant plasmids pBMB1201 and pBMB1202. Both of the mini res fragments, BamHI/HindIII fragment in pBMB1201 and EcoRI/HindIII fragment in pBMB1202, were ligated to the 3.3 kb EcoRI/HindIII fragment of shuttle vector pHT3101, which contained the ori. Ec, ampr and emr antibiotic resistant genes, resulting recombinant plasmid pBMB1203. After deleted the BamHI and EcoRI sites which located ouside the two res sites, resolution vector pBMB1204 was resulted. There are multiple cloning sites between two copies of resolution sites which have the same direction. The plasmid replication origin ori44, which come from B. thuringiensis sub sp. kurstaki strain YBT-1520, was inserted into the multiple cloning sites of pBMB1204 and then resolution shuttle vector pBMB1205 was obtained. With spectinomycin resistant gene as target, it was found that the resolution rate is 100% and the stability of the resolved plasmid is 93%. Using this shuttle vector, antibiotic resistance markers and other non-B. thuringiensis DNA can be selectively eliminated after the selection of transformants by antibiotic resistance marker. This vector is very useful to solve the gene safety problem while has no effect on target gene expression.
Bacillus thuringiensis ; genetics ; DNA Transposable Elements ; Genetic Vectors ; Plasmids ; Replicon

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Study on construct and expression of synthetic genes encoding spider dragline silk in Escherichia coli.

Min LI ; Wen-Xian ZHANG ; Zhi-Hua HUANG ; Jian-Kun HUANG

Chinese Journal of Biotechnology.2002;18(3):331-334.

Dragline spider silk produced from Nephilia clavipes major ampullate is a natural fibrous protein with specific mechanical properties such as high tensile strength and elasticity. Synthetic gene monomer encoding recombinant spider silk protein, based on the known repetitive protein sequence and partial cDNA sequence of dragline silk, was constructed and expressed. DNA monomer sequences were multimerized to encode high molecular weight synthetic spider silks using a "head-to-tail" construction strategy. Multimer was cloned into pET30a(+), a prokaryotic high potency expression vector, and induced with IPTG. The protein from 8-unit repeat was produced in Escherichia coli at levels up to 20 mg/L. The protein was easily purified with high recovery by using an metal ion affinity chromatography and purity was over 90%. The results of SDS-PAGE and Western blot suggested that the mass of the expression product was about 37 kD. This value and amino acid analysis were consistent with those of theoretic calculation.
Amino Acid Sequence ; Amino Acids ; analysis ; Base Sequence ; Escherichia coli ; genetics ; Fibroins ; Molecular Sequence Data ; Molecular Weight ; Proteins ; analysis ; genetics ; isolation & purification ; Recombinant Proteins ; analysis ; isolation & purification

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Construction and in vitro activity of specific dual-ribozyme against alpha 1 (I) and (III) procollagen genes.

Er-Sheng KUANG ; Fu-Hua YANG ; Shu-Juan WEN ; Kai-Jun XIANG

Chinese Journal of Biotechnology.2002;18(3):327-330.

Overexpression of procollagen gene can cause the extraordinary increase of collagen's synthesis and therefore lead to the keloid and hypertrophic scar. To utilize ribozyme to suppress the expression of procollagen genes, a eukaryotic expression recombinant plasmid containing a dual-ribozyme gene against alpha 1 (I) and alpha 1 (III) procollagen genes was constructed. The ribozyme from in vitro transcription was incubated with target transcripts from recombinant plasmids which separately contained the fragments of the second exons of pro alpha 1 (I) and pro alpha 1 (III) collagen genes under various experimental conditions. The results showed that the dual-ribozyme could efficiently catalyze the specific cleavage of the target RNAs at 37 degrees C, 42 degrees C, 50 degrees C and Mg2+ concentration from 10 mmol/L to 20 mmol/L. This work provided a basis for further study on the ribozyme to suppress the expression of procollagen genes and control the cicatrization.
Base Sequence ; Exons ; Molecular Sequence Data ; Procollagen ; genetics ; RNA ; metabolism ; RNA, Catalytic ; genetics ; metabolism ; Temperature

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Callus induction and regeneration from mature seeds of indica rice minghui 63 and anti-fungal assay of transgenic rice plants.

Li-Jiang WANG ; Xiao-Tian MING ; Cheng-Cai AN ; Hua-Yi YUAN ; Zhang-Liang CHEN

Chinese Journal of Biotechnology.2002;18(3):323-326.

A large number of callus from mature seeds of indica rice minghui 63 were obtained through pre-induction on medium with 2 mg/L 2,4-D but without inorganic and organic components for 9 days. Trichosanthin gene was transferred into indica rice minghui 63 by using agrobacterium with the help of bombardment and the transgenic plants were obtained by inducing regeneration. Southern and Western blot analysis showed that the trichosanthin gene had been transferred into genome of minghui 63 and expressed in rice plants. The anti-fungal assay suggested that transgenic rice plants enhanced resistance to infection of Pyricularia oryzae.
Mitosporic Fungi ; drug effects ; Oryza ; genetics ; microbiology ; Plant Diseases ; microbiology ; Plants, Genetically Modified ; Regeneration ; Seeds ; physiology ; Trichosanthin ; genetics

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Study on optimization of expression, purification, properties and biological function of recombinant human sBLyS.

Xiao-Mei YAN ; Shuang-Quan ZHANG ; Da-Peng ZHANG ; Mei-Yan LIU ; Ping LIU

Chinese Journal of Biotechnology.2002;18(3):318-322.

The prokaryotic expression plasmid pET-30a(+)/sBLyS was constructed and transformed into E. coli BL21 (lambda DE3). The recombinant protein was found to be highly expressed by the plight of soluble part and inclusion body. For the sake of enhancing the proportion of the soluble part, inducement at 16 degrees C for 12 h was ascertained. The expressing product was then purified by Ni2+ affinity chromatography gel. PI of the recombinant human sBLyS(rhsBLyS) is about 7.1-7.3 and it assembles into a homotrimer. The effect of rhsBLyS on B lymphocytes by MTT method told us the B lymphocytes' proliferating capacity dose depended on concentration and also stimulating time of the rhsBLys. With rhsBLyS(2 micrograms/mL) stimulating 3 days, B lymphocytes can proliferate the most.
B-Cell Activating Factor ; B-Lymphocytes ; drug effects ; Escherichia coli ; genetics ; Humans ; Isoelectric Point ; Lymphocyte Activation ; drug effects ; Membrane Proteins ; biosynthesis ; isolation & purification ; pharmacology ; Recombinant Proteins ; biosynthesis ; Temperature ; Tumor Necrosis Factor-alpha ; biosynthesis ; isolation & purification ; pharmacology

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Screening for resistance gene candidate from a genomic TAC library of Triticum aestivum-Haynaldia villosa translocation line 6VS/6AL by pooled PCR.

Gen-Ji QIN ; Pei-Du CHEN ; Yao-Guang LIU ; Yu-Da FANG ; Da-Jun LIU

Chinese Journal of Biotechnology.2002;18(3):313-317.

A pair of degenerate primers were designed based on NBS (nucleotide binding site, NBS) domain of resistance(R) gene and used to perform PCR with cDNA from the translocation line 6VS/6AL of Triticum aestivum-Haynaldia villosa. A clone (N7) characterized with NBS was obtained by sequencing analysis. Two specific primers were designed from the N7 sequence and used to screen a genomic TAC (transformation-competent artificial chromosome, TAC) library of 6VS/6AL consisting of ca. 2 x 10(6) clones. The library was stored as clone pools in twenty-two 96-well plates, each well containing approximately 1000 TAC clones. TAC plasmids were prepared from all the 2112 pools. Using a pooled PCR screening procedure, a positive TAC clone having a 40 kb insert was obtained. The positive clone was confirmed by Southern hybridization with the NBS fragment as a probe. The results indicate that the pooled PCR method is effective for screening of genomic libraries having large number of clones.
Amino Acid Sequence ; Base Sequence ; Binding Sites ; Blotting, Southern ; Chromosomes, Artificial ; Genomic Library ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Translocation, Genetic ; Triticum ; genetics

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Construction of eukaryotic expression vector using neomycin-resistance gene mutant as selectable marker.

Chuan GAO ; Xu-Dong ZHU ; Xiao-Wei ZHOU ; Fang YU ; Bai-Song LU ; Pei-Tang HUANG

Chinese Journal of Biotechnology.2002;18(3):308-312.

Neomycin-resistance gene is widely used as a selectable marker in eukaryotic expression vector. It codes neomycin phosphotransferase II (NPT II) which confers resistance to various aminoglycoside antibiotic such as G418 and kanamycine. In this work, by site-directed mutagenesis the neo gene mutant was obtained. The expression vector pmDNA using the neo gene mutant as selectable marker has been constructed. After inserting interest luciferase gene, the expression plasmid pmDNAluc + was stably transfected CHO-K1 cells. As a result, the expression positive ratio reaches to approximate 95% and the ratio of high expression colonies is apparently higher than the controls.
Amino Acid Sequence ; Base Sequence ; Drug Resistance ; genetics ; Genetic Markers ; Genetic Vectors ; Kanamycin Kinase ; genetics ; Molecular Sequence Data ; Mutation

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