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991.
Pamela Osenkowski Hua Li Wenjuan Ye Lorene Aeschbach Michael S. Wolfe Huilin Li 《Journal of molecular biology》2009,385(2):642-652
γ-Secretase, an integral membrane protein complex, catalyzes the intramembrane cleavage of the β-amyloid precursor protein (APP) during the neuronal production of the amyloid β-peptide. As such, the protease has emerged as a key target for developing agents to treat and prevent Alzheimer's disease. Existing biochemical studies conflict on the oligomeric assembly state of the protease complex, and its detailed structure is not known. Here, we report that purified active human γ-secretase in digitonin has a total molecular mass of ∼ 230 kDa when measured by scanning transmission electron microscopy. This result supports a complex that is monomeric for each of the four component proteins. We further report the three-dimensional structure of the γ-secretase complex at 12 Å resolution as obtained by cryoelectron microscopy and single-particle image reconstruction. The structure reveals several domains on the extracellular side, three solvent-accessible low-density cavities, and a potential substrate-binding surface groove in the transmembrane region of the complex. 相似文献
992.
Seiya Mochida Satoshi Tsuzuki Makoto Yasumoto Kuniyo Inouye Tohru Fushiki 《Enzyme and microbial technology》2009,45(4):288-294
Matriptase is a transmembrane serine protease expressed in vertebrates. This enzyme is synthesized as a zymogen form and is converted to an active form by cleavage at the N-terminus of the serine protease catalytic domain. In a mammalian cell-based expression system, we have produced pseudozymogen forms of recombinant matriptase (r-matriptase) that are activated by cleavage with a recombinant enterokinase (r-EK) in vitro. In the present study, four different pseudozymogen forms of r-matriptase containing a site for activation by r-EK and a hexahistidine tag (His6-tag) were expressed in and secreted by Pichia pastoris, a methylotrophic yeast. The pseudozymogens with His6-tag at their C-termini formed multimers linked by intermolecular disulfide bonds. After treatment with r-EK, they exhibited no detectable hydrolytic activity toward a chromogenic substrate. A pseudozymogen form of matriptase catalytic domain with N-terminal His6-tag (designated His6t-S-CD) was secreted as a monomer. His6t-S-CD after r-EK treatment exhibited activity comparable to that of the activated form of an r-matriptase expressed in mammalian cells. His6t-S-CD could be purified from culture medium in milligram quantities. The expression in the yeast offers an efficient method of producing larger amounts of r-matriptase. 相似文献
993.
Diganta Kalita Siva Prasad Das Nashreen S. Islam 《Biological trace element research》2009,128(3):200-219
The present work was undertaken to examine and compare some biologically important properties of peroxo compounds of V(V)
and W(VI) containing biogenic species as ancillary ligand. New anionic peroxovanadate(V) complex of the type Na[VO(O2)2(triglycine)]·3H2O (pV1) and a molecular peroxotungstate(VI) [WO(O2)2(triglycine)]·3H2O (pW1) were synthesized and characterized for the purpose and their stability in solution was ascertained. Studies on kinetics
of inhibition of alkaline phosphatase activity by the newly synthesized compounds and series of dipeptide and amino acid containing
peroxo complexes of vanadium and tungsten synthesized previously by us viz., Na[VO(O2)2(gly-gly)(H2O)]·H2O (gly-gly = glycyl-glycine), Na[VO(O2)2(asn)]·H2O (asn = asparagine), Na[VO(O2)2(gln)]·H2O (gln = glutamine), and [WO(O2)2(gly-gly)(H2O)]·3H2O, revealed that each of these species is a potent mixed-type inhibitor of the enzyme. Significant difference was noted between
the peroxovanadium (pV) and peroxotungsten (pW) compounds in terms of their oxidant activity with reduced glutathione. 相似文献
994.
995.
Shonisani C. Tshidino Jason Krause Abayomi P. Adebiyi Koji Muramoto Ryno J. Naud 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2009,154(2):229-234
A myofibril-bound serine protease (MBSP) was partially purified from ostrich (Struthio camelus) skeletal muscle. MBSP was dissociated from the myofibrillar fraction by ethylene glycol treatment at pH 8.5, followed by partial purification via Toyopearl Super Q 650 S and p-aminobenzamidine column chromatographies. Ostrich MBSP revealed a major protein band of approximately 21 kDa on SDS-PAGE, showing proteolytic activity after casein zymography. Optima pH and temperature of ostrich MBSP were 8 and 40 °C, respectively. Substrate specificity analysis revealed that the enzyme cleaved synthetic fluorogenic substrates at the carboxyl side of arginine residues. Kinetic parameters (Km and Vmax values) were calculated from Lineweaver–Burk plots. The kinetic characteristics of ostrich MBSP were compared to values obtained for commercial bovine trypsin in this study, as well as those obtained for MBSP from mouse and various fish species. The results suggest that ostrich MBSP is a tryptic-like serine protease. Ostrich MBSP exhibited low sequence identity to commercial bovine trypsin (44%), MBSP from lizard fish skeletal muscle (33%) and trypsinogen from ostrich pancreas (22%). 相似文献
996.
Ju Eun Je Sang Jung Ahn Na Young Kim Jung Soo Seo Moo-Sang Kim Nam Gyu Park Joong Kyun Kim Joon Ki Chung Hyung Ho Lee 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2009,154(4):474-485
We assessed the putative physiological roles of cathepsin K from a flatfish, olive flounder. We cloned a cDNA encoding for cathepsin K (PoCtK), a cysteine protease of the papain family from olive flounder, Paralichthys olivaceus. The tissue-specific expression pattern of PoCtK, determined via real-time PCR analysis, revealed ubiquitous expression in normal tissues with high levels of expression in the spleen and bone marrow. However, PoCtK expression was significantly increased in the muscle and gill at 3–24 h post-injection with bacterial lipopolysaccharide (LPS). The cDNA encoding for the mature enzyme of PoCtK was expressed in Escherichia coli using the pGEX-4T-1 expression vector system. Its activity was quantified via the cleavage of the synthetic peptide Z-Gly-Pro-Arg-MCA, zymography, and the collagen degradation assay. The optimum pH for the protease activity was 8, and the recombinant PoCtK enzyme degraded collagen types I, II, III, IV, and VI and acid-soluble collagen from olive flounder muscle in the presence of chondroitin 4-sulphate (C-4S). Therefore, our data indicate that cathepsin K may play a role in the immune system of fish skin and muscle, in addition to its principal bone-specific function as a collagenolytic enzyme. 相似文献
997.
John L. Markley David J. Aceti Craig A. Bingman Brian G. Fox Ronnie O. Frederick Shin-ichi Makino Karl W. Nichols George N. Phillips Jr. John G. Primm Sarata C. Sahu Frank C. Vojtik Brian F. Volkman Russell L. Wrobel Zsolt Zolnai 《Journal of structural and functional genomics》2009,10(2):165-179
The Center for Eukaryotic Structural Genomics (CESG) is a “specialized” or “technology development” center supported by the
Protein Structure Initiative (PSI). CESG’s mission is to develop improved methods for the high-throughput solution of structures
from eukaryotic proteins, with a very strong weighting toward human proteins of biomedical relevance. During the first three
years of PSI-2, CESG selected targets representing 601 proteins from Homo sapiens, 33 from mouse, 10 from rat, 139 from Galdieria sulphuraria, 35 from Arabidopsis thaliana, 96 from Cyanidioschyzon merolae, 80 from Plasmodium falciparum, 24 from yeast, and about 25 from other eukaryotes. Notably, 30% of all structures of human proteins solved by the PSI Centers
were determined at CESG. Whereas eukaryotic proteins generally are considered to be much more challenging targets than prokaryotic
proteins, the technology now in place at CESG yields success rates that are comparable to those of the large production centers
that work primarily on prokaryotic proteins. We describe here the technological innovations that underlie CESG’s platforms
for bioinformatics and laboratory information management, target selection, protein production, and structure determination
by X-ray crystallography or NMR spectroscopy.
相似文献
John L. MarkleyEmail: |
998.
Kristen L. Huber Kevin D. Olson Jeanne A. Hardy 《Protein expression and purification》2009,67(2):139-147
Peptide libraries have proven to be useful in applications such as substrate profiling, drug candidate screening and identifying protein–protein interaction partners. However, issues of fidelity, peptide length, and purity have been encountered when peptide libraries are chemically synthesized. Biochemically produced libraries, on the other hand, circumvent many of these issues due to the fidelity of the protein synthesis machinery. Using thioredoxin as an expression partner, a stably folded peptide scaffold (avian pancreatic polypeptide) and a compatible cleavage site for human rhinovirus 3C protease, we report a method that allows robust expression of a genetically encoded peptide library, which yields peptides of high purity. In addition, we report the use of methodological synchronization, an experimental design created for the production of a library, from initial cloning to peptide characterization, within a 5-week period of time. Total peptide yields ranged from 0.8% to 16%, which corresponds to 2–70 mg of pure peptide. Additionally, no correlation was observed between the ability to be expressed or overall yield of peptide-fusions and the intrinsic chemical characteristics of the peptides, indicating that this system can be used for a wide variety of peptide sequences with a range of chemical characteristics. 相似文献
999.
重组人纤溶酶原基因的酵母表达、产物纯化及鉴定 总被引:1,自引:0,他引:1
目的: 研究重组人纤溶酶原丝氨酸蛋白酶结构域(rhPLG-SP)的酵母表达、纯化及理化性质。 方法:采用7.5 L发酵罐对巴斯德毕赤酵母(Pichia pastoris)工程菌 rhPLG-SP/GS115 进行高密度培养、甲醇诱导表达rhPLG-SP,培养液经三步纯化:超滤、Sephacryl S-100、SP-Sepharose FF,将活性组分透析后冷冻干燥。等点聚焦电泳、HPLC、质谱分别检测 rhPLG-SP等电点、纯度和分子量;纤维蛋白平板、肽底物S-2403 分别测定 rhPLG-SP激活后的纤维蛋白溶解和酰胺水解活性。结果: 7.5 L高密度发酵可获得约为400mg/L培液的表达量,经三步纯化后制备的rhPLG-SP纯度大于96%。理化分析显示 rhPLG-SP的等电点为7.5~7.8,分子量:27 877 Da,比活性:23.6U/mg。结论: 初步建立了rhPLG-SP酵母工程菌的高密度培养、表达及纯化工艺,所制备半成品活性与血浆提取的PLG相近,具备放大生产和应用的潜力。 相似文献
1000.
Leishmania is associated with a broad spectrum of diseases, ranging from simple cutaneous to invasive visceral leishmaniasis. Here, the sequences of ten cysteine proteases of types A, B and C of Leishmania major were obtained from GeneDB database. Prediction of MHC class I epitopes of these cysteine proteases was performed by NetCTL program version 1.2. In addition, by using BcePred server, different structural properties of the proteins were predicted to find out their potential B cell epitopes. According ... 相似文献