Robust microarray production of freshly expressed proteins in a human milieu.
Robust microarray production of freshly expressed proteins in a human milieu.
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DOI：
10.1002/prca.201200063
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发表时间：
2013-06
影响因子：
2
通讯作者：
LaBaer, Joshua
中科院分区：
文献类型：
--
作者：
Festa, Fernanda;Rollins, Sean M.;Vattem, Krishna;Hathaway, Margarita;Lorenz, Phillip;Mendoza, Eliseo A.;Yu, Xiaobo;Qiu, Ji;Kilmer, Greg;Jensen, Penny;Webb, Brian;Ryan, Ed T.;LaBaer, Joshua
体外转录/翻译（IVTT）系统广泛应用于蛋白质组学。对于临床应用，哺乳动物系统对于蛋白质折叠和活性是优选的;然而，获得的蛋白质水平低。从人类细胞中提取的一种新系统（1-Step Human Coupled IVT）有可能克服这一问题，并在人类环境中提供高产率的蛋白质表达。蛋白质印迹和自组装蛋白质微阵列被用来测试蛋白质合成的效率相比，兔网织红细胞裂解物（RRL）的一步人偶联IVT（HCIVT）。该阵列还用于测量从暴露于病原体或疫苗的患者血清中获得的免疫应答。HCIVT在所有实验中的表现均优于RRL。在蛋白质印迹和蛋白质微阵列中，HCIVT中合成的蛋白质产量比RRL高10倍以上。此外，HCIVT显示出稳健的批间重现性。在免疫测定中，许多抗原的信号仅在HCIVT表达的阵列中检测到，这主要是由于背景信号的减少和阵列上蛋白质水平的增加。HCIVT是一种稳健的IVTT系统，可在人体环境中产生高水平的蛋白质。它可用于需要在哺乳动物系统中进行蛋白质表达和高产率的应用。HCIVT表达的蛋白质的免疫原性应答增加对于许多疾病（包括癌症）中的生物标志物发现将是至关重要的。
In vitro transcription/translation (IVTT) systems are widely used in proteomics. For clinical applications, mammalian systems are preferred for protein folding and activity; however, the level of protein obtained is low. A new system extracted from human cells (1-Step Human Coupled IVT) has the potential to overcome this problem and deliver high yields of protein expressed in a human milieu. Western blots and self-assembled protein microarrays were used to test the efficiency of protein synthesis by 1-Step Human Coupled IVT (HCIVT) compared to rabbit reticulocyte lysate (RRL). The arrays were also used to measure the immune response obtained from serum of patients exposed to pathogens or vaccine. HCIVT performed better than RRL in all experiments. The yield of protein synthesized in HCIVT is more than 10 times higher than RRL, in both western blot and protein microarrays. Moreover, HCIVT showed a robust lot-to-lot reproducibility. In immune assays, the signals of many antigens were detected only in HCIVT-expressed arrays, mainly due to the reduction in the background signal and the increased levels of protein on the array. HCIVT is a robust IVTT system that yields high levels of protein produced in a human milieu. It can be used in applications where protein expression in a mammalian system and high yields are needed. The increased immunogenic response of HCIVT-expressed proteins will be critical for biomarker discovery in many diseases, including cancer.
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影响因子：
3.1
作者：
Montor, Wagner R.;Huang, Jin;LaBaer, Joshua
通讯作者：
LaBaer, Joshua
影响因子：
20.3
作者：
Carter, Bing Z.;Qiu, Yi Hua;Kornblau, Steven M.
通讯作者：
Kornblau, Steven M.
DOI：
10.1007/s10969-011-9100-8
发表时间：
2011-07-01
期刊：
Journal of Structural and Functional Genomics
影响因子：
--
作者：
Cormier, Catherine Y.;Park, Jin G.;LaBaer, Joshua
通讯作者：
LaBaer, Joshua
影响因子：
2
作者：
Nnedu, Obinna N.;O'Leary, Michael P.;Vigil, Adam
通讯作者：
Vigil, Adam
影响因子：
--
作者：
Han, Xiaoming;Yamanouchi, Go;Katayama, Yoshiki
通讯作者：
Katayama, Yoshiki