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Heterologous production of bovine plasmin was studied in the industrially relevant bacterium Lactococcus lactis. Two sets of lactococcal gene expression signals were coupled to the region of the plasmin gene coding for the serine protease
domain. When the promoter region of the prtP gene was used, plasmin was detected mainly intracellularly in strain BPL25 by Western blot hybridization. The intracellular
presence of plasmin led to physiological stress. Expression of the plasmin gene driven by the promoter and complete signal
sequence of the lactococcal usp45 gene resulted in efficient plasmin secretion in strain BPL420. Cell lysis was observed in strains producing plasmin fragments
including the catalytic domain, but not in control strains, which only produced a non-catalytic region of plasmin. The plasmin
produced was shown to be biologically active.
Received: 2 December 1996 / Received revision: 17 March 1997 / Accepted: 27 April 1997 相似文献
53.
The effect of water stress on plant water status and net photosynthetic gas exchange (PN) in six barley genotypes (Hordeum vulgare L.) differing in productivity and drought tolerance was studied in a controlled growth chamber. Osmotic adjustment (OA), PN, stomatal conductance (gs), and the ratio intercellular/ambient. CO2 concentration (Ci/Ca) were evaluated at four different levels of soil water availability, corresponding to 75, 35, 25 and 15 % of total available water. Variability in OA capacity was observed between genotypes: the drought tolerant genotypes Albacete and Alpha showed higher OA than drought susceptible genotypes Express and Mogador. The genotype Albacete exhibited also higher PN than the others at low water potential (Ψ). The ratios of PN/gs and Ci/Ca showed that differences in photosynthetic inhibition between genotypes at low Ψ were probably due to nonstomatal effects. In Tichedrett, a landrace genotype with a very extensive root development, OA was not observed, however, it exhibited a capacity to maintain its photosynthetic activity under water stress. 相似文献
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Revision of ploidy status of Dioscorea alata L. (Dioscoreaceae) by cytogenetic and microsatellite segregation analysis 总被引:1,自引:0,他引:1
Gemma Arnau A. Nemorin E. Maledon K. Abraham 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2009,118(7):1239-1249
Dioscorea alata is a polyploid species with several ploidy levels and its basic chromosome number has been considered by most authors to
be x = 10. Standard chromosome counting and flow cytometry analysis were used to determine the chromosome number of 110 D. alata accessions of the CIRAD germplasm collection. The results revealed that 76% of accessions have 2n = 40 chromosomes, 7% have 2n = 60 chromosomes and 17% have 2n = 80 chromosomes. Progenies were produced from 2n = 40 types of D. alata and the segregation patterns of six microsatellite markers in four different progenies were analysed. The Bayesian method
was used to test for diploid versus tetraploid (allo- and autotetraploid) modes of inheritance. The results provided the genetic
evidence to establish the diploidy of plants with 2n = 40 chromosomes and to support the hypothesis that plants with 2n = 40, 60 and 80 chromosomes are diploids, triploids and tetraploids, respectively, and that the basic chromosome number of
D. alata is x = 20. The findings obtained in the present study are significant for effective breeding programs, genetic diversity analysis
and elucidation of the phylogeny and the species origin of D. alata. 相似文献
58.
Guevara T Mallorquí-Fernández N García-Castellanos R García-Piqué S Ebert Petersen G Lauritzen C Pedersen J Arnau J Gomis-Rüth FX Solà M 《Biological chemistry》2006,387(10-11):1479-1486
Cyclisation of N-terminal glutamine and/or glutamate to yield pyroglutamate is an essential posttranslational event affecting a plethora of bioactive peptides and proteins. It is directly linked with pathologies ranging from neurodegenerative diseases to inflammation and several types of cancers. The reaction is catalysed by ubiquitous glutaminyl cyclotransferases (QCs), which present two distinct prototypes. Mammalian QCs are zinc-dependent enzymes with an alpha/beta-hydrolase fold. Here we present the 1.6-A-resolution structure of the other prototype, the plant analogue from Carica papaya (PQC). The hatbox-shaped molecule consists of an unusual five-fold beta-propeller traversed by a central channel, a topology that has hitherto been described only for some sugar-binding proteins and an extracellular nucleotidase. The high resistance of the enzyme to denaturation and proteolytic degradation is explained by its architecture, which is uniquely stabilised by a series of tethering elements that confer rigidity. Strikingly, the N-terminus of PQC specifically interacts with residues around the entrance to the central channel of a symmetry-related molecule, suggesting that this location is the putative active site. Cyclisation would follow a novel general-acid/base working mechanism, pivoting around a strictly conserved glutamate. This study provides a lead structure not only for plant QC orthologues, but also for bacteria, including potential human pathogens causing diphtheria, plague and malaria. 相似文献
59.
Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins 总被引:4,自引:0,他引:4
Affinity tags are highly efficient tools for protein purification. They allow the purification of virtually any protein without any prior knowledge of its biochemical properties. The use of affinity tags has therefore become widespread in several areas of research e.g., high throughput expression studies aimed at finding a biological function to large numbers of yet uncharacterized proteins. In some cases, the presence of the affinity tag in the recombinant protein is unwanted or may represent a disadvantage for the projected application of the protein, like for clinical use. Therefore, an increasing number of approaches are available at present that are designed for the removal of the affinity tag from the recombinant protein. Most of these methods employ recombinant endoproteases that recognize a specific sequence. These process enzymes can subsequently be removed from the process by affinity purification, since they also include a tag. Here, a survey of the most common affinity tags and the current methods for tag removal is presented, with special emphasis on the removal of N-terminal histidine tags using TAGZyme, a system based on exopeptidase cleavage. In the quest to reduce the significant costs associated with protein purification at large scale, relevant aspects involved in the development of downstream processes for pharmaceutical protein production that incorporate a tag removal step are also discussed. A comparison of the yield of standard vs. affinity purification together with an example of tag removal using TAGZyme is also included. 相似文献
60.
Arnau Montagud Emilio Navarro Pedro Fernández de Córdoba Javier F Urchueguía Kiran Raosaheb Patil 《BMC systems biology》2010,4(1):156