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271.
将来源于肠膜明串珠菌Leuconostoc mesenteroides ATCC 12291的蔗糖磷酸化酶(Sucrose phosphorylase,SPase)基因进行密码子优化后将其插入到pET-28a中构建表达载体pET-28a-spase,诱导大肠杆菌Escherichia coli BL21(DE3)/pET-28a-spase表达制得SPase粗酶液,将重组SPase纯化后进行酶学表征。结果表明,重组SPase的比酶活为213.98 U/mg,纯化倍数为1.47倍,酶活回收率达87.80%。该酶最适温度为45℃,最适pH为6.5,该酶对蔗糖的Km为128.8 mmol/L,Vmax为2.167μmol/(mL·min),kcat为39 237.86 min-1,利用重组SPase催化氢醌合成α-熊果苷,最优条件为:氢醌添加量为40 g/L,蔗糖/氢醌的摩尔比为5:1,重组SPase 250 U/mL。在25 mmol/L的吗啉乙磺酸(MES)缓冲液(pH 7.0)中,反应温度30℃,避光反应24 h后终止反应,再用500 U/mL的糖化酶40℃处理2.5 h。α-熊果苷产量达98 g/L,氢醌的转化率接近99%。综上所述,文中克隆并研究了重组SPase,并建立了其在α-熊果苷生产中的应用。 相似文献
272.
目的探究钙结合蛋白S100A16在胰岛素抵抗中的作用。方法用S100A16抗体进行免疫沉淀,然后用蛋白质谱分析寻找与S100A16相互作用的蛋白。实验1转染Vector质粒的HepG2细胞作为对照,用转染shRNA质粒、S100A16过表达质粒干预作为处理组。实验2以慢性胰岛素刺激细胞构建胰岛素抵抗模型,采用转染shRNA质粒的细胞作为对照,用未转染和转染Vector质粒干预作为处理组。实验3以不做任何处理的细胞作为对照,在胰岛素抵抗模型中用吡格列酮干预作为处理组。Western blot检测相关蛋白的表达水平。组间比较采用成组t检验。结果与转染Vector质粒比较,转染S100A16过表达质粒中胎球蛋白A表达(1.39±0.54比2.85±0.25)水平上调(P<0.05);与转染Vector质粒比较,转染shRNA质粒胎球蛋白A蛋白表达(0.36±0.03比0.20±0.03)水平降低(P<0.01)。在胰岛素抵抗条件下,与转染shRNA质粒的细胞比较,未转染和转染Vector质粒的IRS-2蛋白表达(0.11±0.04比1.65±0.48)水平上调(P<0.01);与不做任何处理的细胞比较,用吡格列酮处理的细胞IRS-2表达(0.26±0.11比0.52±0.05)水平上升(P<0.01)。结论S100A16在HepG2细胞中通过胎球蛋白A促进胰岛素抵抗。 相似文献
273.
Joe Carver Domingos Ng Michelle Zhou Peggy Ko Dejin Zhan Mandy Yim David Shaw Brad Snedecor Michael W. Laird Steven Lang Amy Shen Zhilan Hu 《Biotechnology progress》2020,36(4):e2967
Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity. Our results showed that increasing antibody gene copy number can increase specific productivity, but with diminishing returns as more antibody genes are added to the same TI locus. Random integration of additional antibody DNA copies in to a targeted integration cell line showed a further increase in specific productivity, suggesting that targeting additional genomic sites for gene integration may be beneficial. Additionally, the position of antibody genes in the two plasmids was observed to have a strong effect on antibody expression level. These findings shed light on vector design to maximize production of conventional antibodies or tune expression for proper assembly of complex or bispecific antibodies in a TI system. 相似文献
274.
275.
Chen Jing-Jing Shen Jun-Xian Yu Zong-Hao Pan Chuan Han Fei Zhu Xiu-Ling Xu Hui Xu Rui-Ting Wei Tong-Yao Lu Ya-Ping 《Neurochemical research》2021,46(3):660-674
Neurochemical Research - Depression afflicts more than 300 million people worldwide, but there is currently no universally effective drug in clinical practice. In this study, chronic restraint... 相似文献
276.
Wang Han Shen Yi-Jia Li Xiu-Juan Xia Jun Sun Li Xu Yehao Ma Yu Li Dai Xiong Yuan-Chang 《Neurochemical research》2021,46(5):1214-1223
Neurochemical Research - Paclitaxel is a common chemotherapeutic agent in cancer treatment, while it often causes chemotherapy-induced peripheral neuropathy (CIPN), which... 相似文献
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280.
Huaxin Yu Tasuku Hamaguchi Yoshiki Nakajima Koji Kato Keisuke Kawakami Fusamichi Akita Koji Yonekura Jian-Ren Shen 《BBA》2021,1862(10):148471
Photosystem II (PSII) functions mainly as a dimer to catalyze the light energy conversion and water oxidation reactions. However, monomeric PSII also exists and functions in vivo in some cases. The crystal structure of monomeric PSII has been solved at 3.6 Å resolution, but it is still not clear which factors contribute to the formation of the dimer. Here, we solved the structure of PSII monomer at a resolution of 2.78 Å using cryo-electron microscopy (cryo-EM). From our cryo-EM density map, we observed apparent differences in pigments and lipids in the monomer-monomer interface between the PSII monomer and dimer. One β-carotene and two sulfoquinovosyl diacylglycerol (SQDG) molecules are found in the monomer-monomer interface of the dimer structure but not in the present monomer structure, although some SQDG and other lipid molecules are found in the analogous region of the low-resolution crystal structure of the monomer, or cryo-EM structure of an apo-PSII monomer lacking the extrinsic proteins from Synechocystis sp. PCC 6803. In the current monomer structure, a large part of the PsbO subunit was also found to be disordered. These results indicate the importance of the β-carotene, SQDG and PsbO in formation of the PSII dimer. 相似文献