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排序方式: 共有209条查询结果,搜索用时 15 毫秒
11.
用PCR的方法克隆出了编码蓝细菌Synechococcussp.PCC7002FNR中FNR区的基因petHL,克隆到达载体pET3a上,转化大肠杆菌BL21(DE3)后实现了大量表达。重组FNR区(rFNRD)经DEAESephdexA50离子交换层析及SephadexG100凝胶层析得到大量的电泳均一的rFNRD。N末端氨基酸序列分析表明,表达产物确为petHL所编码。且起始Met翻译后未被除去。rFNRD与rFNR的吸收光谱相同,其黄递酶活性的最适pH和最适温度也相同。rFNRD能在体外催化电子从P700到NADP+的传递 相似文献
12.
铁氧还原白-NADP^+氧还酶(FNR)是光合电子传递中的关键酶之一。蓝细菌Synechococcus sp.PCC7002中编码FNR的petH基因被克隆到表达载体pET-3d上,在大肠杆菌中得到了高效表达,其表达量占大肠杆菌总蛋白的505%以上。高效表达的产物以可溶性和包含体两种形式存在。可溶性部分具有FNR的酶活性,在经过离子交换和凝胶层析后产生了电泳纯的FNR。包含体形式的部分经6.7mo 相似文献
13.
We have purified an alkali-tolerant catalase from the thermophilic bacterium Metallosphaera hakonensis. The catalase gene, which encodes 303 amino acids and has a calculated molecular mass of 33 kDa, including its putative signal peptide encoding sequence, was cloned. The deduced amino acid sequence exhibited a region-specific homology with the sequences of manganese catalases from thermophilic bacteria such as Thermus thermophilus and Thermus brockianus. When this gene was overexpressed in Escherichia coli, proteins of the expected size (33 kDa) were overproduced in the inactive form. We made several attempts to obtain active forms of or to activate these overproduced proteins. Upon their induction into E. coli, a 100-fold increase in the catalase activity was detected when high-concentration manganese was used as the medium. The catalase activity of the purified enzyme was optimal at a pH of 10.0. The alkali-tolerant property of this catalase makes it a promising enzyme in biotechnological applications such as H(2)O(2)-detoxifying systems. 相似文献
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Camponova P Baud S Mattras H Duroux-Richard I Bonnafous JC Marie J 《Protein expression and purification》2007,55(2):300-311
The B(2) bradykinin receptor belongs to the G-protein coupled receptor family. Development of new drugs for this important therapeutic target requires structural information on the receptor. The main goal of the present work was to overexpress the human B(2) receptor for future biophysical studies. Different tagged B(2) receptors were engineered and their properties were evaluated by transient expression in HEK293S cells. A B(2) receptor tagged with a hexahistidine at the N-terminus and a nonapeptide at the C-terminus was selected for high expression level and preserved ligand-binding characteristics. First, we generated a HEK293S stable cell line expressing the receptor constitutively at a level of 60pmol/mg of crude membrane protein. However, the decrease of expression level with cell passages led us to express the B(2) receptor in a HEK293S tetracycline-inducible stable cell line. Induction of expression of the B(2) receptor with tetracycline and sodium butyrate led to a level of 100pmol/mg of membrane protein, which is the highest level reported so far for this receptor. The expression level was stable with cell passages and the ligand-binding and signal transduction properties of the receptor were unaltered. The receptor was purified to near homogeneity by solubilization with n-dodecyl-beta-d-maltoside followed by a two-step purification procedure combining hydroxyapatite and immunoaffinity chromatography. Although the purified receptor is not functional, the purification of the B(2) receptor to near homogeneity from a stable cell line overexpressing this receptor pave the way for future structural studies of this receptor. 相似文献
16.
Nakagawa T Ikehata R Myoda T Miyaji T Tomizuka N 《Protein expression and purification》2007,54(2):295-299
Cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2 was overexpressed in Escherichia coli using the Cold expression system and the recombinant enzyme, rBglAp, was characterized. The purified rBglAp exhibited similar enzymatic properties to the native enzyme, e.g., (i) it had high activity at 0 degrees C, (ii) its optimum temperature and pH were 10 degrees C and 8.0, respectively, and (iii) it was possible to rapidly inactivate the rBglAp at 50 degrees C in 5 min. Moreover, rBglAp was able to hydrolyze both ONPG and lactose with K(m) values of 2.7 and 42.1mM, respectively, at 10 degrees C. One U of rBglAp could hydrolyze about 70% of the lactose in 1 ml of milk in 24h, and the enzyme produced trisaccharide from lactose. We conclude that rBglAp is a cold-active enzyme that is extremely heat labile and has significant potential application to the food industry. 相似文献
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角毛壳菌几丁质酶基因chi58多点突变及在毕赤酵母中的高效表达 总被引:1,自引:0,他引:1
应用重叠延伸PCR技术(gene splicing by overlap extension PCR,gene SOEing),简称SOE-PCR对角毛壳菌(Chaetomium cupreum)的几丁质酶基因chi58进行多点突变.依据毕赤酵母密码子偏爱性,将毕赤酵母中编码Arg使用频率几乎为0的密码子CGC突变为使用频率高的AGA,构建了含有正确突变的酵母表达载体pPIC9K-chi58A,电转化毕赤酵母GS115,获得的重组酵母株在诱导120 h酶活力最高,平均可达101.71 U/mL±3.33 U/mL;其活力比未优化重组酵母株(31.83 U/mL±4.85 U/mL)提高了约3倍,且经10代传代培养后遗传稳定性良好.表达产物的SDS-PAGE分析表明,酶蛋白分子大小为58 kD. 相似文献
20.
Khan F Jala VR Rao NA Savithri HS 《Biochemical and biophysical research communications》2003,306(4):1083-1088
Diaminopropionate ammonia-lyase gene from Escherichia coli and Salmonella typhimurium was cloned and the overexpressed enzymes were purified to homogeneity. The k(cat) values, determined for the recombinant enzymes with DL-DAP, D-serine, and L-serine as substrates, showed that the enzyme from S. typhimurium was more active than that from E. coli and the K(m) values were found to be similar. The purified enzymes had an absorption maximum (lambda(max)) at 412 nm, typical of PLP dependent enzymes. A red shift in lambda(max) was observed immediately after the addition of 10mM DL-DAP, which returned to the original lambda(max) of 412 nm in about 4 min. This red shift might reflect the formation of an external aldimine and/or other transient intermediates of the reaction. The apoenzyme of E. coli and S. typhimurium prepared by treatment with L-cysteine could be partially (60%) reconstituted by the addition of PLP. The holo, apo, and the reconstituted enzymes were shown to be present as homo dimers by size exclusion chromatography. 相似文献