全文获取类型
收费全文 | 1218篇 |
免费 | 78篇 |
国内免费 | 1篇 |
专业分类
1297篇 |
出版年
2023年 | 4篇 |
2022年 | 5篇 |
2021年 | 13篇 |
2020年 | 5篇 |
2019年 | 17篇 |
2018年 | 18篇 |
2017年 | 17篇 |
2016年 | 31篇 |
2015年 | 36篇 |
2014年 | 57篇 |
2013年 | 61篇 |
2012年 | 86篇 |
2011年 | 70篇 |
2010年 | 53篇 |
2009年 | 50篇 |
2008年 | 82篇 |
2007年 | 76篇 |
2006年 | 72篇 |
2005年 | 65篇 |
2004年 | 65篇 |
2003年 | 67篇 |
2002年 | 68篇 |
2001年 | 10篇 |
2000年 | 15篇 |
1999年 | 13篇 |
1998年 | 17篇 |
1997年 | 22篇 |
1996年 | 20篇 |
1995年 | 27篇 |
1994年 | 12篇 |
1993年 | 11篇 |
1992年 | 16篇 |
1991年 | 7篇 |
1990年 | 11篇 |
1989年 | 8篇 |
1988年 | 6篇 |
1987年 | 7篇 |
1986年 | 5篇 |
1985年 | 4篇 |
1984年 | 6篇 |
1982年 | 10篇 |
1981年 | 4篇 |
1980年 | 7篇 |
1978年 | 3篇 |
1977年 | 3篇 |
1976年 | 3篇 |
1973年 | 4篇 |
1972年 | 7篇 |
1971年 | 3篇 |
1970年 | 3篇 |
排序方式: 共有1297条查询结果,搜索用时 0 毫秒
71.
72.
The aromatase cytochrome P-450 and its clinical impact 总被引:8,自引:0,他引:8
73.
High-level expression and rapid purification of rare-codon genes from hyperthermophilic archaea by the GST gene fusion system 总被引:5,自引:0,他引:5
Wu X Oppermann U 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,786(1-2):177-185
In this study, we compared two gene fusion expression strategies using two rare codon genes (Ssh10b and MtGrxM) from archaea as a model system. Both genes can be highly expressed as N- or C-terminal fusion partners to GST or the intein/chitin-binding tag. However, the fusion protein with intein tag could not be cleaved, even under stringent conditions, possibly due to steric hindrance, thus preventing further purification. In contrast, the GST fusion system could increase protein expression level and the corresponding fusion protein could be easily cleaved by thrombin. After binding to glutathione sepharose, the fusion protein was cleaved on column, and a roughly purified protein fraction was eluted. This fraction was purified by heating at 80 degrees C for 10 min, followed by centrifugation. The correct total mass and N-terminal primary structure were confirmed by mass spectrometry and Edman degradation. Both constructs were used for in vitro expression, and similar results were obtained, indicating higher expression levels of the GST tag vs. intein/chitin tag. Taken together, our results suggest that the GST fusion system can be used as a considerable alternative to synthetic genes for the expression of rare codon genes. The affinity chromatography purification followed by a heating step is an efficient and convenient method for thermostable protein purification. 相似文献
74.
75.
Udo H Lam CK Mori S Inouye M Inouye S 《Journal of molecular microbiology and biotechnology》2000,2(4):557-563
Eukaryotic cells contain a large number of protein Ser/ Thr kinases, which play important roles in signal transduction required for cell proliferation, differentiation, and stress response and adaptation. It is also known that some prokaryotes contain a family of protein Ser/Thr kinases. A major challenge in the characterization of these kinases is how to identify their specific substrates. Here we developed such a method using a protein Ser/Thr kinase, Pkn2 from Myxococcus xanthus, a Gram-negative soil bacterium. When Pkn2 is inducibly expressed in E. coli, cells are unable to form colonies on agar plates. This lethal effect of Pkn2 was eliminated in an inactive Pkn2 mutant in which the highly conserved Lys residue was changed to Asn, indicating that phosphorylation of a cellular protein(s) in E. coli resulted in growth arrest. Several clones from an E. coli genomic library were found to suppress the lethal effect when co-expressed with pkn2. Four out of seven multi-copy suppressors were identified to encode HU, (3 for HUalpha and 1 for HUB) a histone-like DNA binding protein. Purified HUalpha was found to be specifically phosphorylated by Pkn2 at Thr-59, and the phosphorylated HUalpha became unable to bind to DNA, suggesting that the phosphorylation of endogenous HU proteins by Pkn2 contributed at least in part to the lethal effect in E. coli. The present method termed the STEK method (Suppressors of Toxic Effects of Kinases) may be widely used for the substrate identification not only for prokaryotic protein Ser/Thr kinases but also for eukaryotic kinases. 相似文献
76.
A 25.9-kb plasmid, pXU5, encoding high level cadmium resistance was isolated from Staphylococcus aureus strain ATCC25923. A labelled cadA probe from plasmid pI258 hybridised to a 2.3-kb EcoRI fragment of pXU5. pXU5 was incompatible with an S. aureus incompatibility group 1 plasmid. 相似文献
77.
Walter E. Horton Jr. Imelda Udo Patricia Precht Richard Blakir Karen Hasty 《In vitro cellular & developmental biology. Animal》1998,34(5):378-384
Summary The objective of this study was to determine if an immortalized mammalian chondrocyte cell line had a profile of matrix metalloproteinase
(MMP) expression that was consistent with what has been reported for primary chondrocytes in vitro and in vivo. A combination of zymography, Western, and Northern analysis was used to examine the expression of MMPs that are relevant
to cartilage degradation. Both interleukin-1β and tumor necrosis factor α induced a 4- to 9-fold increase in the level of
MMP-9 expression in conditioned media, and a 17- to 24-fold increase in MMP-3 mRNA. Other compounds such as basic fibroblast
growth factor and staurosporine each increased MMP-9 expression individually and potentiated the effects of the two cytokines.
Transforming growth factor β had no positive or inhibitory effects. N-methyl arginine blocked the increase in nitric oxide observed following treatment with the cytokines but did not prevent
the increased expression of MMPs. The pattern of metalloproteinase expression observed in IRC cells and the response to cytokines
is very similar to what has been reported during the pathogenesis of osteoarthritis. The IRC cells should be useful as a model
system to study basic mechanisms controlling chondrocyte MMP expression and to identify pharmacological modulators of this
process. 相似文献
78.
79.
Molecular dynamics simulations using AMB06C, an in-house carbohydrate force field, (NPT ensembles, 1 atm) were carried out on a periodic cell that contained a cyclic 240 glucose residue amylose fragment (c-DP-240) and TIP3P water molecules. Molecular conformation and movement of the amylose fragment and water molecules at different temperatures were examined. The periodic cell volume, density, and potential energy were determined at temperatures above and below the glass transition temperature (Tg) in 25 K increments. The amorphous cell is constructed through successive dynamic equilibration steps at temperatures above the assumed Tg value and the temperature successively lowered until several temperature points were obtained below Tg. Molecular dynamics simulations were continued for at least 500 ps or until the volume drift stopped and remained constant for several hundred picoseconds. The Tg values were found by noting the discontinuity in slope of the volume (V), potential energy (PE), or density (ρ) versus 1/T. The changes in flexibility and motion of the amylose chain as well as differences in self diffusion coefficients of water molecules are described. The final average Tg value found (316 K) is in agreement with experimental values, i.e. 320 K. 相似文献
80.
A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five ‘pQLink’ vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expressed individually; to achieve co-expression, two pQLink plasmids are combined by ligation-independent cloning. pQLink co-expression plasmids can accept an unrestricted number of genes. As an example, the co-expression of a heterotetrameric human transport protein particle (TRAPP) complex from a single plasmid, its isolation and analysis of its stoichiometry are shown. pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags. We demonstrate pull-down experiments of human valosin-containing protein (VCP) with fragments of the autocrine motility factor receptor (AMFR). The cloning method avoids PCR or gel isolation of restriction fragments, and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid. It is expected that applications are not restricted to bacteria, but could include co-expression in other hosts such as Bacluovirus/insect cells. 相似文献