The methylotrophic yeast, Pichia pastoris, has been one of most widely used expression system for heterologous proteins production in the past decades. In this review, we mainly focus on the influences of methanol utilization phenotypes (i.e Mut+, MutS and Mut-) and gene dosage on recombinant protein expression in genetically engineered Pichia pastoris. Strains with different phenotype not only exhibit different methanol utilization rate and growth rate, but also differ in terms of foreign protein production and byproducts production. Gene dosage has a complex impact on heterologous protein expression. Foreign gene copy number could positively correlate to protein production, whereas the negative correlation often occurs especially when gene dosage goes high. The latter might be related with post-translational processing of target protein like disulfide bond formation, folding, etc and some studies show that protein production yield can be enhanced by co-expression of proteins with chaperon activity, like PDI(protein disulfide isomerise). Physiologic profiles of strains with different gene dosage were also compared.
To construct a scFv library by phage display technique from the spleen cells of mice immunized with B3HM cells. Three mice were immunized with B3HM cells, and their spleen cells were harvested. The genes of VH and Vk were amplified by RT-PCR from the cDNA of the immunized spleen cells and a scFv-phage display antibody library was constructed. The capacity of library was measured,and the variety of the library was analyzed by digesting with restriction endonuclease BstNI..ScFv phage clones were randomly picked and identified phage-scFv clone by binding B3HM cells using immunofluorescein. We produce a scFv library containing 5×106 individual clones which showed different patterns after digested with restriction endonuclease BstNI. Individnal phage-scFv clone showed B3HM cells positive using immunofluorescein. A scFv library of anti-B3HM cell surface molecules has been constructed. It will be useful for finding out some novel genes of causing leukemia, and establishs the infarctate foundation of clarifying the pathogenesis of leukemiagenesis.
In order to construct a prokaryotic expression vector of human receptor syt II N-fragment and to express recombinant MBP-Syt fusion protein in E.coli and to purify and identify its activity. According to codon preference of E.coli, a DNA fragment encoding human syt II N-fragment was synthesized, and then cloned into prokaryotic vector pMAL-c2x for sequencing. Then the recombinant plasmid pMAL-Syt was introduced into E.coli ER2566 by transformation for expression and the obtained engineered bacteria were induced by IPTG. The fusion protein was purified by amylose resin affinity chromatography and identified by SDS-PAGE and Western blot. The binding activity of the protein was determined by ELISA. It is concluded that MBP-Syt protein is of good binding activity.
To investigate the biological role of snake venom cystatin(sv-cystatin) in tumor progression, the cDNA of sv-cystatin amplified by PCR from pUC18-cystatin plasmid was cloned into methanol-inducible expression vector pPICZαA. The linearized recombinant palsmid pPICZαA-cystatin was transfered into Pichia pastoris, strain GS115 by electrophoration. Transfermants with phenotype Mut＋ selected were identified by PCR analysis and induced in 1.0% methanol. The reombinant sv-cystatin protein was examined by SDS-PAGE, Western blot analysis. The molecular mass of expression product was about 14 KD and approximately 16 mg/L of recombinant sv-cystatin was produced from one of GS115-cystatin transformants. The chromatography purified protein could reduce the activity of papain. The ability of B16F1 cells treated with recombinant sv-cystatin to invade the reconstituted basement membrane decreased significantly (P<0.01). Our data provides the first evidence that sv-cystatin could not only inhibit papain activity, but also suppress the Matrigel invasion of B16F1 Melanoma cell line.
The Bcl-2 family of proteins play a central role in the control of apoptosis, a fundamental process for both human health and disease, by mitochondrial pathway. PUMA(p53 up-regulated modulator of apoptosis protein) is one of BH3-only members of Bcl-2 family , its function is to promote cell apoptosis. To obtain BH3 death domain peptide of PUMA and detect its biological activity, the synthesized double-stranded oligomeric nucleotide encoding PUMA-BH3 peptide was cloned into expression vector pTYB2，thus generating a construct of pTYB2-PUMA-BH3 which expressed PUMA-BH3-intein-chitin binding domain fusion protein. Then the recombinant plasmid was transformed into E.coli BL-21 (DE3) and fusion protein was expressed under induction by IPTG. The soluble PUMA-BH3 peptide was purified from chitin affinity chromatography by DTT reduction. Through measuring mitochondria viability(MTT),mitochondria permeability transition(MPT) and the translocation of cytochrome c(Cyt c ) assayed by western blotting, the biological pro-apoptotic activity of PUMA-BH3 peptide was studied. The PUMA-BH3 peptide has the effects on decreasing the mitochondria viability remarkably , inducing mitochondrial swelling and promoting Cyt c releasing from isolated mitochodria . Mitochondrial swelling and the release of Cyt c induced by PUMA-BH3 peptide concerned with the opening of MPT，which can be improved by cyclosporine A(CsA).These results indicated that recombinant PUMA-BH3 peptide might possess pro-apoptosis activity and paved a reasonable way for the study of new apoptosis regulators.
The known members of inhibitor of growth (ING) gene family are considered as candidate tumor suppressor genes. ING4, a novel member of ING family, is recently reported to regulate brain tumour angiogenesis through transcriptional repression of NF-kB-responsive genes, induce G2/M arrest by the increased p21 expression in a p53-dependent manner, suppress the loss of contact inhibition and represses activation of the hypoxia inducible factor, which plays an important role in the progression of tumorigenesis. However, seldom studies about ING4 inducing tumor cells apoptosis were reported. In our research, the C6 cells (mouse glioma cells) were infected respectively with the blank adenovirus carrying GFP (Ad) and the recombinated Ad-hING4-His (constructed by our department), then RT-PCR assay was used to detect the transcriptions of hING4, as well Western-blotting assay was ued to detect the expressions of hING4. The effects of hING4 expression upon C6 cells were observed, and the growth curve was drawed and tumor control rates were calculated. The C6 cells, which were affected by blank Ad and Ad-hING4-His, were respectively observed by LSCM (laser scan confocal microscope) and transmission electron microscope (TEM), detected by flow cytometry; and the genomic DNA of both groups were extracted and electrophoresised in agarose gel to examinate the DNA fragments. The results showed hING4 can significantly inhibit the growth of C6 cells by promoting the cell's apoptosis, which probably is the first one to prove this property of ING4. This research established the experimental and theoretical foundation for gene therapy for gliomas with ING4 in the future.
The role of conserved insulin residues TyrB26 was studied to better understand the relationship between insulin and its receptor. Insulin analogues with a single amino acid substitution or single N-methylation of the peptide bond in the position B26 were all shortened in the C-terminus of the B-chain by four amino acids. The effect of modifications was followed by the binding to the human insulin receptor. From our results: [HistidineB26]-des-tetrapeptide-(B27-B30)-insulin -B26-amide and [N-MeHisB26]-des-tetrapeptide-(B27-B30)-insulin-B26-amide had no significant effect on the binding affinity and they showed binding affinity 72 and 107 % of that of human insulin respectively; [N-MeGluB26]-des-tetrapeptide-(B27-B30)-insulin-B26-amide, [AadB26]-des- tetrapeptide-(B27-B30)-insulin-B26-amide and [Phe(4-carboxyB26)]-des-tetrapeptide-(B27-B30)– insulin-B26-amide affected the potency highly positively in vitro studies; they showed binding affinity 130, 234, 160%, respectively, of that of human insulin.
Objective: To investigate effects of bFGF and collagen on tissue-engineered cartilage in pellet culture. Methods: Growth-plate chondrocytes were incubated in pellet culture. bFGF and Collagen was applied alone or together based on the results of previous research.The histological structure of neocartilage was observed by stained with H＆E and Immunohistochemical analysis. The DNA quantity of neocartilage was measured by hoechst33258 dye.The production of matrix was estimated based on hydroxyproline quantity and Alcian Blue stain method. Results: The histological structure of in vitro-formed tissue was similar to growth-plate cartilage.The DNA quantity of neocartilage increased by the supplement of bFGF and collagen. The production of matrix was promoted by collagen. bFGF and collagen suppressed the expression of collagen Ⅰ,and decelerated the decrease of collagenⅡ. The combined usage of bFGF and collagen had synergistic effects. Conclusion: Pellet culture maintained the phenotype of chondrocyte and promoted matrix production. bFGF and Collagen improved the construction of engineered cartilage. The combined usage of bFGF and collagen had synergistic effects on the regeneration of cartilage.
The polysaccharide from Porphyra yezoensis（PYP） was purified and analyzed by biochemical techniques. The effect of PEP on the proliferation of MCF-7 was investigated in vitro. Methods: The purified polysaccharide from Porphyra yezoensis was obtained by DEAE-cellulose and Sephadex G-200 column chromatography. The effect of PEP on the proliferation of MCF-7 was investigated in vitro by MTT method. Two components, PY-D1 and PY-D2, were isolated from the Porphyra yezoensis polysaccharide by ion-exchange chromatography DEAE-52. Two polysaccharides, PY-G1 and PY-G2, were further isolated from PY-D2 Ueda by gel filtration Sephadex G-200. PY-G was identified by UV and IR spectra. All of components could inhibit the proliferation of MCF-7. Results: It was found polysaccharides of Porphyra yezoensis had antitumor activity for MCF-7 cells.
To identify specific protein markers for gastric cancer detection and diagnosis, as well as develop new potential therapeutic targets of the disease. Gastric cancer tissues and adjacent normal mucosa were examined by the fluorescence differential in-gel electrophoresis (DIGE) technique after labeled with CyDye DIGE fluors Cy3, Cy5 and Cy2. Protein spots detected differential with statistical significance were identified by MALDI-TOF MS or MS/MS. As the result, intensity changes of 33 spots were detected with statistical significance. 9 protein spots of them up-regulated otherwise 24 down-regulated in gastric cancer tissues. And 22 of them were identified by MALDI-TOF MS or MS/MS successfully. Several proteins up-regulated such as MnSOD, HSP60, mutant desmin et al. HSP27, prostaglandin F synthase, SeBP 1, zinc finger protein 160, tubulin alpha 6, eTEF1 A 1 and so on were down-regulated. Our results suggest that DIGE is a useful technique for differential expressed proteins screening and analysis in gastric cancer tissues which may be useful for the development of new molecular markers for diagnosis and prognosis of gastric carcinoma. This differently expressed proteins may be useful tumor markers for gastric cancer.
During the dilute acid pretreatment of lignocellulosic materials such as corn stover, hemicellulose is hydrolyzed into monosaccharides, and meanwhile, toxic by-products are simultaneously generated, which may influence ethanol fermentation thereafter. Studies on the inhibitory effects of the by-products on ethanol fermentation are of practical use for further improvement of ethanol yield from lignocellulosic materials. Five by-products, including acetic acid, formic acid, vanillin, furfural and 5-hydroxymethylfurfural, were identified to be the main components in the hydrolysate of dilute acid pretreatment of local corn stover, which were added into the medium at different concentrations to study their impacts on the growth and ethanol fermentation of a recombinant xylose-utilizing yeast strain, S. cerevisiae 6508-127. The ethanol production was inhibited by formic acid and acetic acid to a lesser extent than that to the growth, and formic acid was shown to be much more toxic than acetic acid, showing severe inhibitory effects at the concentration of 1g/L, half of the concentration for acetic acid which showed remarkably negative effects on ethanol fermentation. Vanillin caused a much longer lag-phase in growth when the concentration was 2g/L, and the lag-phase was not obvious at lower concentrations. At the concentration of 6g/L, vanillin completely inhibited the fermentation as well as the cell growth. 5-Hydroxymethylfurfural was showed to remarkably inhibit ethanol production, but the biomass yield was higher by exogenous addition of 5-Hydroxymethylfurfural than control. Furfural at 0.5-1.5g/L inhibited the cell growth, but the ethanol yield was higher than that of the control experiment. It was also found that vanillin, furfural and 5-hydroxymethylfurfural could be assimilated and metabolized by S. cerevisiae 6508-127 under the experimental conditions.
Criteria had been established for the preliminary evaluation of plant species as potential biodiesel raw materials based on the major specifications of biodiesel standards of Germany, European Organization and USA. Fatty acid composition, Iodine Value (IV) and cetane number (CN) were commonly considered to predict the quality of fatty acid methyl esters of seed oils. We established four criteria to screen oil-bearing trees in China in this article, including: 51
The influence of separating effect of different chromatographic conditions of mouse gut flora by high performance ion exchange chromatography analysis was studied. The optimum chromatographic conditions for separating gut bacteria were determined. The sample was applied to the chromatography column packed with Toyopearl SuperQ-650c anion resin, equilibrated with 0.02mol/L piperazin-hydrochloric acid buffer (pH 8.0), and elution salt 1mol/L NaCl, eluted with the gradient of 0-50% NaCl/ 80 min, then 50-75% NaCl/ 25 min at the flow rate 1mL/min, and injecting volume was 1mL.Under these conditions, intestinal flora were separated into several fractions. The establishment of HPLC analysis method will lay a foundation of further research on the components of mouse gut flora and their dynamic changes.
Potato cultivar Atlantic is widely grown for potato chips in the world. However, this economically important potato cultivar exhibits very poor yields and traits under severe environmental stress. To develop an efficient plant transformation system that could be used to produce large scale transgenic potato plants with enhanced tolerance to environmental stress and therefore would be beneficial for potato processing industry, Agrobacterium-mediated transformation of internodal stem explants using both superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes under the control of an oxidative stress-inducible SWPA2 promoter was performed in this study. Comparing to leaf explants, stem internodal explants were less liable to damage during manipulation, more amenable to in vitro conditions. The addition of silver thiosulfate to the selection medium considerably promoted the shoot induction from explant-derived callus. Seven to nine shoots per stem explant were obtained. By combining the best treatments, this system yielded shoot induction frequency of 94.2% and transformation frequency of 80% of internodal stem explants. Stable integration of the transgenes was confirmed by PCR and Southern blot analyses. In conclusion, Short duration (7-8 weeks), high efficiency and easy process make this system well suited for wider commercial applications of transgenic Atlantic potato plants.
Human sTNFR1 (soluble tumor necrosis factor receptor 1) gene was amplified by RT-PCR from Hela cells. A recombinant expression vector of sTNFR1-MBP was constructed in pMAL-c2x, and transformed into E. Coli JM109.It was sequenced and confirmed to be identifical to the sTNFR1 gene in data bank. Recombinant protein sTNFR1-MBP was induced by IPTG and purified by Amylose resin Affinity Chromatography. sTNFR1-MBP was binded to sTNFR1's antibody in Western-blotting. From MTT assays, the results showed that sTNFR1-MBP could effectively block the cytotoxicity mediated by TNFαon QSG7701 cells. Annexin V-FITC staining and flowcytometry were used to observe the recombinant protein's anti-apoptosis capacity and the recombinant protein has marked anti-apoptosis effect in vitro.sTNFR1-MBP had good biological activity and it will be employed in further study.
We used Uniform Design to optimize some relative factors influencing the expressing of t-PA variant-rPA(K) in E.coli system, and found the best expressive conditions. They were(by using 300 mL culture fask): the volume of media was 25 mL, inducing time was 5.3 hours, pH was 6.0, the concentration of IPTG was 0.1 mmol/L, inducing time was 25?C, and best culture media was HD. After being optimized, the yield of expression had been improved from 0.16 to 0.48, and it was as 3 times as before. The results above will offer the basement for purification and renaturation of rPA(K).
Objective: To identify all the potential ORFs encoding secreted proteins in Escherichia coli UTI89 genome, and to preliminarily characterise the secreted proteins and the signal peptides. Methods: Entire 5211 ORFs were predicted by network softwares including SignalP3.0, TatP1.0, SecretomeP2.0, etc. The basic features of signal peptides and secreted proteins in prediction results were statistically analysed. The secreted proteins which have the same signal sequence were aligned by the programe Blast 2 Sequences. Results: 432 ORFs encoding Sec pathway secreted proteins, 19 ORFs encoding Tat pathway secreted proteins and 386 ORFs encoding non-classically secreted proteins exist in E. coli UTI89 genome. The mean length of signal peptides is 25.5 aa, and the mean length of secreted proteins is 282.8 aa. The top three frequent amino acids in signal peptides are L, A, S. Only two signal peptides have the same sequence, and the corresponding secreted proteins are highly homologous. Conclusions: 837 ORFs in E.coli UTI89 genome may encode secreted proteins. Lengths of most secreted proteins are less than 500 aa. Amino acids in signal peptides are relatively conservative, most of which are hydrophobic. Signal peptides diversify widely but the secreted proteins containing the same signal sequence are likely to be encoded by homologous genes.
Microsystems give new platforms for cell research. Cell patterning technology, which is an absolutely new way of cell culturing, plays an important role in the field of “cells on chip”. Here, we reviewed the main strategies of cell patterning, including photolithography, soft lithography, stencil-assisted and so on. Furthermore, we introduced the main uses of the microsystems with cell patterning technology, such as fundamental studies in cell biology, tissue engineering and cell-based biosensors.
As the development of mechanization and transportation，there are more and more peripheral nerve wound happened. Although the damaged nerves can be bridged by an end to end surgery operation, when the damnification is very long, the surgery would loss its effect because of existing of stress between the two ends. Nerve guide conduit can provide a proper micro-environment for the self-regeneration of damaged nerve. This paper introduces the judge criterion of nerve self-regeneration, the development of nerve guide conduits materials, the effect of nerve generated factors， some preparation methods of nerve guide conduits， and on the base of foregoing researches, present the required properties of materials and structures for appropriate nerve guide conduit.
With the fast development of biopharmacy,transgenic chicken oviduct bioreactor is going to be the focus of people's attention.For its excellent advantages,transgenic chicken will be one of rising industries in the field of biopharmacy and hot spot of science study.Now,the most successful method for producing transgenic chicken is mediated by retrovirus transferring gene into it, and transgenic chicken have been produced by this method.The review mainly summarizes the advantages and methods of producing transgenic chicken by retrovirus,and the progress in transgenic chicken research.The significance and existent problems of transgenic chicken are also discussed.
The growth and development of plant is affected by environmental factors that include light,temperature and water. Many cis-elements that are response to these elicitors interact with transcription factors, and regulate the expression of target genes. This paper concludes by pointing out recent studies of cis-elements and transcription factors in the plant environmental response promoters, such as light, temperature and water inducible promoter. The molecular mechanisms of gene expression regulated by environmental factors was discussed. This is important to study the mechanisms that plant adapt the environment.
Rare sugars were defined as monosaccharides and their derivatives that rarely exist in nature. They played an important role in food, health, medicine and etc.. A strategy for bioproduction of rare sugars, namely Izumoring, was reviewed in this paper. By the Izumoring method, all monosaccharides and polyols could be linked using enzymatic reaction with D-tagatose 3-epimerase, aldose isomerases and polyol dehydrogenases. According to this strategy, the Izumoring for hexoses, pentoses and tetroses were designed respectively, and the bioproduction routes of various rare sugars, using microbial and enzymatic reactions, could be obtained.
Due to the depletion of fossil energy, bioenergy has become more and more important and the patents in this field increased rapidly in recent years. Patent information is expanding as a unique source of technical information in analyzing technology innovation, development, and strategic planning. The patent databases on the Internet and special software, offer the opportunity to patent bibliometric research. Using Derwent Innovation Index, this article analyzes the patents in 3 key bioenergy technical areas: biological hydrogen, bioethanol and biodiesel, during 1996~2006, from the following aspects: temporal and spatial distributions, patents citations, major fields, influential patent assignees, to reflect the trend and development of key bioenergy techniques.
