共查询到20条相似文献,搜索用时 31 毫秒
1.
Andriy Y. Voronovsky Olha V. Rohulya Charles A. Abbas Andriy A. Sibirny 《Metabolic engineering》2009,11(4-5):234-242
The thermotolerant yeast Hansenula polymorpha ferments glucose and xylose to ethanol at high temperatures. However, H. polymorpha cannot utilize starchy materials or xylans. Heterologous amylolytic and xylanolytic enzymes have to be expressed in this yeast to provide for utilization and growth on starch and xylan. Genes SWA2 and GAM1 from the yeast Schwanniomyces occidentalis, encoding α-amylase and glucoamylase, respectively, were expressed in H. polymorpha. The expression was achieved by integration of the SWA2 and GAM1 genes under the strong constitutive promoter of the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase gene (HpGAP) into H. polymorpha genome. Resulting transformants acquired the ability to grow on a minimal medium containing soluble starch as a sole carbon source. Ethanol production at high-temperature fermentation from starch by the recombinant strains was up to 10 g/L. The XYN2 gene encoding endoxylanase of the fungus Trichoderma reseei was expressed in H. polymorpha. Co-expression of xlnD gene coding for β-xylosidase of the fungus Aspergillus niger and the XYN2 gene in H. polymorpha was achieved by integration of these genes under control of the HpGAP promoter. Resulting transformants were capable of growth and alcoholic fermentation on a minimal medium supplemented with birchwood xylan as a sole carbon source at 48 °C. 相似文献
2.
Mitali Samaddar James F. Catterall Rajan R. Dighe 《Protein expression and purification》1997,10(3):345-355
Follicle-stimulating hormone (FSH), a pituitary gonadotropin, is a heterodimer composed of an α subunit, which is common to all the glycoprotein hormones, noncovalently associated with the hormone-specific β subunit. The objective of the present study is to develop a recombinant DNA expression system for the β subunit of FSH that can be applied to study structure–function relationships while producing large quantities of the hormone subunit for immunocontraceptive, clinical, and veterinary purposes. We report here the expression of biologically active bovine FSHβ (bFSHβ) in the methylotrophic yeastPichia pastoris.ThePichia-expressed FSHβ (pFSHβ) was secreted into the culture medium and was found to be immunologically very similar to pituitary-derived ovine FSHβ. Replacement of the cognate signal peptide with the yeast α mating factor signal peptide increased the level of expression from 230 ng/ml (cognate signal peptide) to 4 μg/ml (α mating factor signal peptide) of the culture supernatant. pFSHβHis.tag(pFSHβ with six histidine residues at the C terminus) was purified to apparent homogeneity using one-step nickel affinity chromatography. The molecular weight of purified pFSHβHis.tagwas approximately 22,000, which was slightly higher than that of the pituitary-derived ovine FSHβ. pFSHβHis.tagcould assemble with the α subunit to yield a heterodimer capable of binding to the FSH receptors and also elicit biological response. These data show that pFSHβHis.tagis properly folded and biologically active. 相似文献
3.
Huo X Liu Y Wang X Ouyang P Niu Z Shi Y Qiu B 《Protein expression and purification》2007,54(2):234-239
Bovine follicle-stimulating hormone (bFSH) is a pituitary gonadotropin composed of two non-covalently associated polypeptide subunits, which must be glycosylated, folded, and assembled as a heterodimer to be biologically active. Low-level expression of the recombinant bFSH is the factor that limits its usefulness as a superovulation treatment for cows. To increase the production of recombinant bFSH, human protein disulfide isomerase (hPDI) was expressed simultaneously in engineered Pichia strains. The secretion characteristics of bFSH with or without hPDI were examined. The co-expression of bFSH and hPDI is increased to 1.56 mg/l of heterodimer in the culture medium, which is 6-fold higher when compared with the control strain carrying the bFSH gene only. These results may be generally applicable to increase the expression of other glycoprotein hormones in yeast. 相似文献
4.
Laminin (LM)-332 (α3β3γ2), a large heterotrimeric glycoprotein, is an essential component of epithelial basement membranes that promotes cell adhesion and migration. Here, we expressed human LM-332 using a novel protein expression system based on the trypanosomatid protozoan host Leishmania tarentolae. Plasmids containing cDNA encoding full-length β3 and γ2 subunits and truncated α3 subunit were sequentially introduced into L. tarentolae. A recombinant strain harboring the three subunits of human LM-332 efficiently formed heterotrimer and secreted it into the culture medium. Heterotrimeric recombinant LM-332 (rLM-332) could be purified from culture medium with one-step immuno-affinity chromatography. The eluted fraction contained all three subunits, as confirmed by immunoprecipitation and immunoblotting. The purified rLM-332 showed similar cell adhesion activity to rLM-332 purified from mammalian cells, indicating its proper folding and assembly. The obtained expression level was not high; however, we suggest that this expression system has the potential for mass production of LMs for tissue engineering. 相似文献
5.
6.
Minho Cha Eun Jung Kim Jihyang Park Juhan Kim Byung-Gee Kim 《Enzyme and microbial technology》2008,43(7):480-485
Various yeast strains were examined for the microbial reduction of ethyl-3-oxo-3-phenylpropanoate (OPPE) to ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE), which is the chiral intermediate for the synthesis of a serotonin uptake inhibitor, Fluoxetine. Kluyveromyces lactis KCTC 7133 was found as the most efficient strain in terms of high yield (83% at 50 mM) and high optical purity ee > 99% of S-HPPE. Based on the protein purification, activity analysis and the genomic analysis, a fatty acid synthase (FAS) was identified as the responsible β-ketoreductase. To increase the productivity, a recombinant Pichia pastoris GS115 over-expressing FAS2 (α-subunit of FAS) of K. lactis KCTC7133 was constructed. In the optimized media condition, the recombinant P. pastoris functionally over-expressed the FAS2. Recombinant P. pastoris showed 2.3-fold higher reductase activity compared with wild type P. pastoris. With the recombinant P. pastoris, the 91% yield of S-HPPE was achieved at 50 mM OPPE maintaining the high optical purity of the product (ee > 99%). 相似文献
7.
Comparison of two expression platforms in respect to protein yield and quality: Pichia pastoris versus Pichia angusta 总被引:1,自引:1,他引:0
Matthias Mack Marion Wannemacher Birgit Hobl Patricia Pietschmann Bjrn Hock 《Protein expression and purification》2009,66(2):165-171
The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor (MTF). Both recombinant proteins were engineered to contain an N-terminal Strep- (WSHPQFEK) and a C-terminal His6-tag. In addition, both proteins contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion. Following vector construction, transformation and zeocin amplification, the best Pichia producers were identified in a screening procedure using Western blot and a Luminex xMAP™ based high-throughput method. Recombinant NK1-fragment and MTF were purified from culture supernatants of the best producers by affinity chromatography (Ni–nitrilotriacetic acid columns). Using P. pastoris as a host for the synthesis of NK1-fragment a protein yield of 5.7 mg/l was achieved. In comparable expression experiments P. angusta yielded 1.6 mg/l of NK1-fragment. NK1-fragment apparently was not glycosylated in either system. For the production of MTF, P. pastoris was also the superior host yielding 1.2 mg/l glycosylated recombinant protein whereas P. angusta was clearly less efficient (<0.2 mg/l MTF). For both expression systems no correlation between the amount of recombinant protein and the copy number of the chromosomally integrated heterologous genes was found. In P. pastoris strains less degradation of the two model recombinant proteins was observed. Altogether, this paper provides a structured protocol for rapidly identifying productive Pichia strains for the synthesis of full-length recombinant proteins. 相似文献
8.
《Phytochemistry》2002,60(8)
Two apiose-containing kaempferol triosides, together with nine known flavonoids were isolated from the leaves of Silphium perfoliatum L. Their structures were elucidated by acid hydrolysis and spectroscopic methods including UV, LSI MS, FAB MS, CI MS, 1H, 13C and 2D-NMR, DEPT, HMQC and HMBC experiments. The two new compounds were identified as kaempferol 3-O-β-
-apiofuranoside 7-O-α-
-rhamnosyl-(1′→6)-O-β-
-galactopyranoside and kaempferol 3-O-β-
-apiofuranoside 7-O-α-
-rhamnosyl-(1→ 6)-O-β-
(2-O-E-caffeoylgalactopyranoside). 相似文献
9.
10.
The large family of signal transducing proteins known as G proteins are heterotrimers that dissociate into an independent α-subunit and βγ-subunit complex after ligand binding or other stimulation. For Gα, at least 30 distinct sequences representing 10 different classes have been identified. On the other hand, cDNAs for only three Gβ-subunit genes have been isolated so far. All three of the Gβ genes have been chromosomally mapped in the human, but only two in the mouse. Using a human retinal cDNA for the third G protein β-subunit, we have mapped the corresponding gene, termed Gnb-3, to mouse Chromosome 6 with somatic cell hybrids and have positioned it distal to but near the marker Raf-1 by analysis of the progeny of three genetic crosses. 相似文献
11.
K. Christopher Min Stephen A. Gravina Thomas P. Sakmar 《Protein expression and purification》2000,20(3):514
For reconstitution studies with rhodopsin and cGMP phosphodiesterase (PDE), all three subunits of heterotrimeric transducin (Tαβγ) were simultaneously expressed in Sf9 cells at high levels using a baculovirus expression system and purified to homogeneity. Light-activated rhodopsin catalyzed the loading of purified recombinant Tα with GTPγS. In vitro reconstitution of rhodopsin, recombinant transducin, and PDE in detergent solution resulted in cGMP hydrolysis upon illumination, demonstrating that recombinant transducin was able to activate PDE. The rate of cGMP hydrolysis by PDE as a function of GTPγS-loaded recombinant transducin (T*) concentration gave a Hill coefficient of approximately 2, suggesting that the activation of PDE by T* was cooperatively regulated. Furthermore, the kinetic rate constants for the activation of PDE by T* suggested that only the complex of PDE with two T* molecules, PDE · T2*, was significantly catalytically active under the conditions of the assay. We conclude that the model of essential coactivation best describes the activation of PDE by T* in a reconstituted vertebrate visual cascade using recombinant heterotrimeric transducin. 相似文献
12.
Ken Horii Takashi Adachi Tetsuya Matsuda Tsutomu Tanaka Hiroshi Sahara Seiji Shibasaki Chiaki Ogino Yoji Hata Mitsuyoshi Ueda Akihiko Kondo 《Journal of Molecular Catalysis .B, Enzymatic》2009,59(4):297-301
β-Glucosidase (BGL1) from Aspergillus oryzae was efficiently produced in recombinant A. oryzae using sodM promoter-mediated expression system. The yield of BGL1 was 960 mg/l in liquid culture, which is 20-fold higher than the yield of BGL1 produced using the yeast Saccharomyces cerevisiae. Recombinant BGL1 converted isoflavone glycosides into isoflavone aglycones more efficiently than β-glucosidase from almond. In addition, BGL1 produced isoflavone aglycones even in the presence of the insoluble form of isoflavone glycosides. 相似文献
13.
14.
A genomic fragment encoding αAPC and βAPC (i.e., α and β units of the allophycocyanin, APC) from Anacystis nidulans UTEX 625 was cloned and sequenced. This fragment, containing a non-coding sequence of 56 nucleotides in between, was then subcloned into the expression vector pMal-c2 downstream from and in frame with the malE gene of E. coli encoding MBP (maltose binding protein). The fusion protein was purified by amylose affinity chromatography and cleaved by coagulation factor Xa. αAPC and βAPC were then separated from MBP and MBP fusion proteins, respectively, and concentrated by membrane centrifugation. The study provides a method to produce recombinant allophycocyanin subunits for biomedical and biotechnological applications. 相似文献
15.
Four xanthone O-glycosides, polygalaxanthones IV–VII were isolated from the roots of Polygala tenuifolia Willd., together with eight known compounds. The structures of the four xanthone O-glycosides were established as 6-O-[α-
-rhamnopyranosyl-(1→2)-β-
-glucopyranosyl]-1-hydroxy-3,7-dimethoxyxanthone (polygalaxanthone IV), 6-O-[α-
-rhamnopyranosyl-(1→2)-β-
-glucopyranosyl]-1,3-dihydroxy-7-methoxyxanthone (polygalaxanthone V), 6-O-(β-
-glucopyranosyl)-1,2,3,7-tetramethoxyxanthone (polygalaxanthone VI), and 3-O-[α-
-rhamnopyranosyl-(1→2)-β-
-glucopyranosyl]-1,6-dihydroxy-2,7-dimethoxyxanthone (polygalaxanthone VII), respectively, on the basis of analysis of spectroscopic evidence. 相似文献
16.
Anna Stochmal Iwona Kowalska Bogdan Janda Angela Perrone Sonia Piacente Wiesaw Oleszek 《Phytochemistry》2009,70(10):1272-1276
Three phenolic glycosides 5-O-{[5′′-O-E-(4′′′-O-threo-guaiacylglycerol)-feruloyl]-β-apiofuranosyl-(1→2)-β-xylopyranosyl} gentisic acid, 5-O-[(5′′-O-vanilloyl)-β-apiofuranosyl-(1→2)-β-xylopyranosyl] gentisic acid and 1-O-[E-(4′′′-O-threo-guaiacylglycerol)-feruloyl]-3-O-β-galacturonopyranosyl glycerol were isolated and identified from the roots of Medicago truncatula together with four known 5-O-β-xylopyranosyl gentisic acid, vicenin-2, hovetrichoside C and pterosupin identified for the first time in this species. Structural elucidation was carried out on the basis of UV, mass, 1H and 13C NMR spectral data. 相似文献
17.
Hichem Henchiri Bernard Bodo Alexandre Deville Lionel Dubost Lazhar Zourgui Aly Raies Philippe Grellier Lengo Mambu 《Phytochemistry》2009,70(11-12):1435-1441
An antiplasmodial bioguided investigation of the EtOAc extract of the aerial parts of Teucrium ramosissimum led to isolation and identification of three sesquiterpenoids, teucmosin, 4α-hydroxy-homalomenol C, 1β,4β,7α-trihydroxy-8,9-eudesmene and two trinorsesquiterpenoids, 4β-hydroxy-11,12,13-trinor-5-eudesmen-1,7-dione and 1β,4β-dihydroxy-11,12,13-trinor-8,9-eudesmen-7-one together with five known sesquiterpenoids, oplopanone, homalomenol C, oxo-T-cadinol, 1β,4β,6β-trihydroxyeudesmane, 1β,4β,7α-trihydroxyeudesmane and four flavonoids, 5-hydroxy-7,4′-dimethoxyflavone, salvigenin, genkwanin and cirsimaritin. The structures and the relative stereochemistry were elucidated by extensive spectroscopic studies including 1D and 2D NMR and mass spectrometry (MS). Homalomenol C, 4β-hydroxy-11,12,13-trinor-5-eudesmen-1,7-dione, oxo-T-cadinol and 1β,4β,6β-trihydroxyeudesmane displayed a significant in vitro antiplasmodial activity against Plasmodium falciparum with IC50 values ranging from 1.2 to 5.0 μg/ml. Furthermore, no cytotoxicity was observed upon the human diploid lung cell line MRC-5 for these compounds. 相似文献
18.
Rainer Roggenkamp Hans Hansen Michael Eckart Zbigniew Janowicz Cornelis P. Hollenberg 《Molecular & general genetics : MGG》1986,202(2):302-308
Summary A high frequency transformation system for the methylotrophic yeast Hansenula polymorpha has been developed. This system depends on complementation of isolated uracil auxotrophs by the URA3 gene of Saccharomyces cerevisiae. Maintenance of the uracil prototrophy is based on integration of plasmid YIp5 at random sites within the H. polymorpha genome and on autonomously replicating plasmids containing ARS1 of S. cerevisiae or related sequences cloned from the host DNA. The sequence of one autonomously replicating sequence (HARS1) from H. polymorpha has been determined showing an AT-rich region of 9 bp with some similarity to the consensus sequence of known eukaryotic replication origins. Mitotic loss of autonomously replicating sequences is high; selection for stable uracil prototrophs yields multiple tandem arrangement of the transformed DNA with no detectable loss of the phenotype on non-selective medium. These features offer the possibility for extensive gene expression in H. polymorpha. 相似文献
19.
Pneumocystis carinii (PC) is an exclusively extracellular pathogen which causes pneumonia in immunocompromised individuals. Histologic studies have demonstrated that PC organisms attach preferentially to type I alveolar epithelial cells and rarely bind to type II cells. Previous reports have demonstrated that cultured type II cells develop a type I cell-like phenotype and express type I cell surface antigens. The current study examines the attachment of PC organisms to isolated rat type H alveolar epithelial cells as a function of time in culture. PC attachment to isolated type II cells increased as the type II cells differentiated in culture from 2.3 ± 1.2% on Day 2 to 18.4 ± 2.7% by Day 8. Previous studies have indicated a role for fibronectin (Fn) and Fn receptors as mediators of PC attachment. Addition of anti-Fn antibodies decreased attachment of PC to Day 8 type II cells from 19.4 ± 2.5% to 9.4 ± 1.9% (P < 0.01). Addition of antibodies to the αv and α5 integrin subunits resulted in significant decreases in PC attachment to Day 8 type II cells. Examination of expression of αv and α5 integrins on Day 2 and Day 8 type II cells demonstrated increased expression of both αv and α5 integrin subunits on Day 8 type II cells. Overall these data indicate that attachment of PC to isolated rat type II cells increases as the cells differentiate into a type I cell-like phenotype in vitro and correlates with increased expression of Fn-binding integrins on the cell surface of the cultured type II cells. 相似文献
20.
Kahali B Trott R Paroush Z Allada R Bishop CP Bidwai AP 《Biochemical and biophysical research communications》2008,373(4):637-642
Hairy is a repressor that regulates bristle patterning, and its loss elicits ectopic bristles (neural hyperplasia). However, it has remained unknown whether Hairy is regulated by phosphorylation. We describe here the interaction of protein kinase CK2 and Hairy. Hairy is robustly phosphorylated by the CK2-holoenzyme (CK2-HoloE) purified from Drosophila embryos, but weakly by the catalytic CK2α-subunit alone, suggesting that this interaction requires the regulatory CK2β-subunit. Consistent with this, Hairy preferentially forms a direct complex with CK2-HoloE. Importantly, we demonstrate genetic interactions between CK2 and hairy (h). Thus, flies trans-heterozygous for alleles of CK2α and h display neural hyperplasia akin to homozygous hypomorphic h alleles. In addition, we show that similar phenotypes are elicited in wild-type flies upon expression of RNAi constructs against CK2α/β, and that these defects are sensitive to h gene dosage. Together, these studies suggest that CK2 contributes to repression by Hairy. 相似文献